WO2023130855A1 - 一种高产率胶原海绵的制备工艺 - Google Patents

一种高产率胶原海绵的制备工艺 Download PDF

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WO2023130855A1
WO2023130855A1 PCT/CN2022/134658 CN2022134658W WO2023130855A1 WO 2023130855 A1 WO2023130855 A1 WO 2023130855A1 CN 2022134658 W CN2022134658 W CN 2022134658W WO 2023130855 A1 WO2023130855 A1 WO 2023130855A1
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tissue
solution
collagen
add
sodium
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French (fr)
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孙冰冰
张娈
张伟
张伊骁
宋默微
刘利
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天新福（北京）医疗器材股份有限公司
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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/10—Other agents for modifying properties
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2201/00—Foams characterised by the foaming process
    • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
    • C08J2201/048—Elimination of a frozen liquid phase
    • C08J2201/0482—Elimination of a frozen liquid phase the liquid phase being organic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the invention relates to a collagen fiber, in particular to a preparation process for a high-yield collagen sponge used in medical devices.
  • Collagen is one of the main components of the extracellular matrix, accounting for about 1/3 of the total protein mass of mammals. It is the main component of connective tissues or organs such as skin, ligaments, cartilage, and tendons. Collagen has unique physical and chemical properties and excellent Biocompatibility, degradability, low immunity and other advantages make it easy to be absorbed in the body, strong hydrophilicity, non-toxic and safe, and become one of the choices of biomedical materials. Generally, it is rich in collagen fibers Extraction of animal tissues such as skin, tendons, bones, etc. Collagen extraction methods usually include alkali method, salt method, acid method, and enzymatic method. According to its solubility, collagen can be divided into: collagen fibers, acid-soluble collagen, and water-soluble collagen.
  • Chinese patent document CN101569765A relates to a type I medical collagen material that maintains the unique triple helix structure of collagen and its extraction method, which is used in tissue repair. At the same time, the material has a certain tensile strength, reduces tissue adhesion, and is beneficial to tissue repair. and regeneration. Its scheme is as follows:
  • Bovine Achilles tendon tissue is frozen and cut into thin slices of 0.1-3 mm, soaked in 0.1% sodium dihydrogen phosphate/sodium hydroxide solution for 1-3 hours under stirring, and kept in neutral conditions.
  • Enzyme reaction add 1:5 (w/w) fig enzyme to the above solution for full reaction, stir for 4 hours, then react with 1% ammonium nitrate for 6 hours, and wash.
  • Salting out add 1M sodium chloride to treat and control the temperature, adjust the pH, stand overnight at 4°C, take it out and react with 2M sodium hydroxide solution, and then neutralize the buffer with 50% sulfuric acid to make the solution slightly acidic. Remove the precipitate.
  • Freeze-drying and film formation dissolve the material obtained above in 0.01-0.5M glacial acetic acid to obtain a 0.5%-2% solution, add 0.05% in 1-8h at 15000-30000rad/min, 0-15°C -60% aminodextran, made into a composite suspension, after freeze-drying, the chain connection between collagen molecules is strengthened by physical or chemical methods, sterilized by low-temperature ethylene oxide, and the pores are made at 160- A membrane with a three-dimensional pore structure between 350 ⁇ m.
  • the suspension obtained by the above method cannot uniformly disperse the components in a good manner, and has low tensile strength and fast degradation rate.
  • the present invention provides a high-yield collagen fiber preparation process, which does not destroy collagen during the preparation process. Structure, after a series of conditional treatments, the final product is collagen fibers, and the collagen sponge prepared by collagen fibers has higher tensile strength, burst strength and longer degradation cycle.
  • the purpose of the present invention is to provide a high-yield collagen fiber preparation process.
  • the preparation process comprises the following steps:
  • Pre-treatment soak the tissue pieces or tissue blocks obtained in step (1) in sodium chloride solution, the concentration of sodium chloride solution is 0.5-1.5wt%, magnetic stirring is carried out for the first time mixing, and then Washing to remove the sodium chloride solution; then adding ethanol solution to the soaked tissue, the concentration of the ethanol solution is 60-90wt%, magnetic stirring for the second mixing, and then washing to remove the ethanol solution to form a pre-treated tissue;
  • Enzyme reaction in an acidic or slightly alkaline solution, add 10-20wt% of the pretreated tissue obtained in step (2), then add protease for enzymolysis, stop the reaction after the enzymolysis is completed; then wash to remove the solution , forming a tissue to be pulverized;
  • Pulverizing pulverizing the tissue to be pulverized obtained in step (3) and then sieving to form pulverized animal tissue;
  • Alkali treatment In the solution containing sodium hydroxide and sodium sulfate, add 10-20wt% of the pulverized animal tissue obtained in step (4) to react; after the reaction is completed, use acid to neutralize to a pH of 4.0 -5.0, then wash with water to form collagen that maintains a triple helical structure;
  • step (3) ficin, pepsin or trypsin can be used for enzymatic hydrolysis, and multiple tests have shown that enzymolysis of the pretreated tissue with ficin has a better enzymatic hydrolysis effect and higher purity of the obtained collagen.
  • step (3) the protease is ficin, and in the acetic acid-sodium acetate buffer solution with a pH of 5.5-6.8, 10-20 wt% of the pre-treated tissue is added, and then 0.1 wt% of the pre-treated tissue is added.
  • -2wt% ficin react with magnetic stirring at 25-37°C for 0.5-5h, after the reaction is completed, add sodium chlorite, ammonium nitrate, hydrogen peroxide or a combination of them, and react at 25°C-37°C for 0.5 -2h, stop the enzyme reaction, wash to remove the solution, and form the tissue to be pulverized.
  • the protease is pepsin
  • an acidic solution with a pH of 2.5-4.5 10-20 wt% of the pre-treated tissue is added, and then 1.0-15 wt% of the pre-treated tissue is added with pepsin , at 25-37°C, react for 2-12h, after the reaction is completed, add 0.5-10M sodium hydroxide to neutralize the solution until the pH is 5.0, stop the enzyme reaction, wash to remove the solution, and form a tissue to be pulverized.
  • a concentrated alkali solution (10M sodium hydroxide)
  • a dilute alkali solution 0.5M sodium hydroxide
  • step (3) the protease is trypsin, in a weakly alkaline solution with a pH of 7.5-7.8, 10-20 wt% of the pre-treated tissue is added, and then 1.0-15 wt% of the pre-treated tissue is added Trypsin, react at 25-37°C for 2-12 hours, after the reaction is completed, add 1M hydrochloric acid solution to neutralize the solution until the pH is 5.0, stop the enzyme reaction, wash to remove the solution, and form the tissue to be pulverized.
  • high temperature is used to kill enzymes to terminate the enzyme reaction.
  • the collagen will be denatured.
  • the change of pH value is not enough to terminate the ficin reaction, therefore, adding one or more combinations of sodium chlorite, ammonium nitrate, and hydrogen peroxide is more effective for terminating the enzyme reaction.
  • step (3) if only sodium chlorite is added to the enzymolysis tissue, the amount of sodium chlorite added is 0.05-0.5wt% of the pretreatment tissue; if only sodium chlorite is added to the enzymolysis tissue If ammonium nitrate is added, the amount of ammonium nitrate added is 1-10wt% of the pretreated tissue; if only hydrogen peroxide is added to the enzymatic tissue, the amount of hydrogen peroxide added is 1-10wt% of the pretreated tissue %.
  • the enzymatic process effectively removes elastin.
  • the other two alternatives of the enzymatic reaction stage also effectively remove elastin without disrupting the structure of collagen fibers.
  • the tissue rich in collagen of the animal is bovine or pig skin, Achilles tendon.
  • the collagen-rich tissue of the animal is cut into 0.3-1.5 mm thick slices or 0.3-1.0 mm cubes.
  • step (2) the tissue sheet or tissue piece is soaked in 0.5-1.5% sodium chloride solution, and the ratio of adding 5-10ml of sodium chloride solution per gram of tissue is mixed for the first time , magnetically stirred at 25-37°C for 12-36h, then washed with water to remove the sodium chloride solution; adding 5-10ml of ethanol solution with a concentration of 60-90wt% per gram of soaked tissue for the second mixing, Stir magnetically at 25-37°C for 12-36h, wash with water to remove the ethanol solution, and form a pre-treated tissue.
  • the particle size of the pulverized animal tissue is 0-300 ⁇ m.
  • step (5) in the mixed solution containing 1.0-2.0M sodium hydroxide and 1.0-2.0M sodium sulfate, add 10-20wt% of the pulverized animal tissue, and react at 25-37°C for 36 -72h; then neutralize with acid to pH 4.0-5.0, and then wash with water to form collagen that maintains a triple helix structure.
  • the alkali treatment stage can effectively remove the telopeptide and obtain virus-inactivated tissue.
  • the concentration of sodium hydroxide in the mixed solution is 1.0-2.0M, and the concentration of sodium sulfate is 1.0-2.0M.
  • the acid used for neutralization is hydrochloric acid or sulfuric acid; the concentration of the acid used is 0.5-10M.
  • step (6) in the 0.05M acetic acid solution, the collagen and sodium chondroitin sulfate obtained in step (5) are added to homogenize, and the collagen that maintains the triple helix structure is homogenized.
  • the addition amount is 0.5wt% of the acetic acid solution, and the addition amount of the sodium chondroitin sulfate is 0.05wt% of the acetic acid solution; homogenize for 2 hours with a homogenizer, and freeze-dry the solution obtained after homogenization to prepare into a collagen sponge.
  • the triple helix structure-maintaining collagen with a purity of up to 99% prepared in step (5) was taken, and 0.05% sodium chondroitin sulfate was added, and mixed in a 0.05M acetic acid solution with a homogenizer for 2 hours.
  • the resulting solution is poured into a medical stainless steel freezer, and the collagen sponge is made by means of low-temperature freezing-condensation-sublimation-heating freeze-drying.
  • the role of acetic acid in the present invention the collagen swells under acidic conditions so that it can be uniformly dispersed after homogenization, and the acetic acid can be completely volatilized in the subsequent freeze-drying process.
  • sodium chondroitin sulfate is the main functional component of the extracellular matrix, which itself also has cell interaction, and covalently bonds with collagen to enhance the ability to induce tissue regeneration. Improve product mechanics and anti-degradation properties.
  • the steps of freeze-drying include in sequence:
  • Low-temperature freezing stage the temperature is -40°C, and the time is 350 minutes;
  • Vacuum drying stage temperature is -18°C, 720min, vacuum degree is 0.2bar;
  • the first drying stage the temperature is -12°C, 400min, and the vacuum is 0.2bar;
  • the second drying stage the temperature is 14°C, 400min, and the vacuum is 0.2bar;
  • the third drying stage the temperature is 0°C, 90min, and the vacuum is 0.2bar;
  • the fourth drying stage the temperature is 25°C, 90min, and the vacuum is 0.2bar.
  • the present invention has the following beneficial effects:
  • the present invention On the premise of removing telopeptides, elastin and inactivating viruses, the present invention has higher extraction efficiency, thoroughly removes miscellaneous proteins, and the prepared collagen has higher purity.
  • the weight ratio of animal tissue or collagen of the present invention accounts for the solution determines the usage amount of process water and the scale of the reaction device, and the minimum mass ratio in all steps of the present invention is nearly 10 times higher than that of other acid/enzyme extraction methods , can greatly save the production space.
  • the collagen prepared by the present invention has higher purity and lower miscellaneous protein content ( ⁇ 0.25%).
  • the collagen sponge prepared by the invention Compared with the acid-dissolved collagen sponge, the collagen sponge prepared by the invention has higher tensile strength, burst strength and longer degradation period.
  • Fig. 1 shows the SDS-PAGE pattern of collagen according to the specific example 3 of the present invention; wherein line1, line3, line5 are acid soluble collagen; line2, line4, line6 are enzymatically hydrolyzed collagen by collagenase; line7 is collagenase aqueous solution.
  • ficins used in the following examples are plant-extracted proteases purchased from MP Biomedicals LLC; enzyme activity ⁇ 200 Bapa U/g.
  • the preparation process of the high-yield collagen fibers comprises the following steps:
  • Pre-treatment soak the tissue pieces or tissue blocks obtained in step (1) in sodium chloride solution, the concentration of sodium chloride solution is 0.5-1.5wt%, magnetic stirring is carried out for the first time mixing, and then Washing to remove the sodium chloride solution, washing to form soaked tissue; adding ethanol solution to the soaked tissue, the concentration of the ethanol solution is 60-90wt%, magnetic stirring for the second mixing, and then washing to remove the ethanol solution , forming a pretreatment organization;
  • Enzyme reaction in an acidic or slightly alkaline solution, add 10-20wt% of the pretreated tissue obtained in step (2), then add protease for enzymolysis, stop the reaction after the enzymolysis is completed; then wash to remove the solution , forming a tissue to be pulverized;
  • Alkali treatment In the solution containing sodium hydroxide and sodium sulfate, add 10-20wt% of the pulverized animal tissue obtained in step (4) to react; after the reaction is completed, use acid to neutralize to a pH of 4.0- 5.0, and then wash with water to form collagen that maintains the triple helical structure;
  • step (3) the protease is ficin, and in the acetic acid-sodium acetate buffer solution with a pH of 5.5-6.8, 10-20 wt% of the pre-treated tissue is added, and then 0.1 wt% of the pre-treated tissue is added.
  • -2wt% ficin react with magnetic stirring at 25-37°C for 0.5-5h, after the reaction is completed, add sodium chlorite, ammonium nitrate, hydrogen peroxide or a combination of them, and react at 25°C-37°C for 0.5 -2h, stop the enzyme reaction, wash to remove the solution, and form the tissue to be pulverized.
  • the protease is pepsin
  • an acidic solution with a pH of 2.5-4.5 10-20 wt% of the pre-treated tissue is added, and then 1.0-15 wt% of the pre-treated tissue is added with pepsin , at 25-37°C, react for 2-12h, after the reaction is completed, add 0.5-10M sodium hydroxide to neutralize the solution until the pH is 5.0, stop the enzyme reaction, wash to remove the solution, and form a tissue to be pulverized.
  • step (3) the protease is trypsin, in a weakly alkaline solution with a pH of 7.5-7.8, 10-20 wt% of the pre-treated tissue is added, and then 1.0-15 wt% of the pre-treated tissue is added Trypsin, react at 25-37°C for 2-12 hours, after the reaction is completed, add 0.5-10M hydrochloric acid to neutralize the solution until the pH is 5.0, stop the enzyme reaction, wash to remove the solution, and form the tissue to be pulverized.
  • step (3) if only sodium chlorite is added to the enzymolysis tissue, the amount of sodium chlorite added is 0.05-0.5wt% of the pretreatment tissue; if only sodium chlorite is added to the enzymolysis tissue If ammonium nitrate is added, the amount of ammonium nitrate added is 1-10wt% of the pretreated tissue; if only hydrogen peroxide is added to the enzymatic tissue, the amount of hydrogen peroxide added is 1-10wt% of the pretreated tissue %.
  • the collagen-rich tissue of the animal is bovine or pig skin, Achilles tendon; preferably, the collagen-rich tissue of the animal is cut into 0.3-1.5 mm thick slices or 0.3 - 1.0mm cubic nubs.
  • step (2) the tissue sheet or tissue piece is soaked in 0.5-1.5% sodium chloride solution, and the ratio of adding 5-10ml of sodium chloride solution per gram of tissue is mixed for the first time , magnetically stirred at 25-37°C for 12-36h, then washed with water to remove the sodium chloride solution; adding 5-10ml of ethanol solution with a concentration of 60-90wt% per gram of soaked tissue for the second mixing, Stir magnetically at 25-37°C for 12-36h, wash with water to remove the ethanol solution, and form a pre-treated tissue.
  • the particle size of the pulverized animal tissue is 0-300 ⁇ m.
  • step (5) in the mixed solution containing 1.0-2.0M sodium hydroxide and 1.0-2.0M sodium sulfate, add 10-20wt% of the pulverized animal tissue, and react at 25-37°C for 36 -72h; then neutralize with acid to pH 4.0-5.0, and then wash with water to form collagen that maintains a triple helix structure.
  • the acid used for neutralization is hydrochloric acid or sulfuric acid; the concentration of the acid used is 0.5-10M.
  • step (6) add 0.05wt% sodium chondroitin sulfate to the collagen that maintains the triple helix structure, mix evenly, add to 0.05M acetic acid solution, and use a homogenizer to homogenize for 2 hours. The resulting solution was freeze-dried to make a collagen sponge.
  • the present embodiment provides a kind of preparation technology of high-yield collagen fiber, and described preparation technology comprises the following steps:
  • Pre-treatment soak the tissue piece obtained in step (1) in a sodium chloride solution with a concentration of 1.5%, mix it with magnetic stirring for the first time, then wash it with water to remove the sodium chloride solution, and pour it into the soaked tissue Add ethanol solution to the mixture, the concentration of the ethanol solution is 90wt%, magnetic stirring is carried out for the second mixing, and then the ethanol solution is removed by washing with water to form a pre-treated tissue;
  • This embodiment provides a collagen fiber with high yield, and the preparation process includes the following steps:
  • pre-treatment soak the tissue piece obtained in step (1) in a sodium chloride solution with a concentration of 1.5%, and mix for the first time at a ratio of adding 10ml of sodium chloride solution per gram of tissue, Stir magnetically at 25-37°C for 36 hours, then wash to remove the sodium chloride solution; add 5ml of ethanol solution with a concentration of 60wt% per gram of soaked tissue for the second mixing, and stir magnetically at 25-37°C After 12 hours, wash with water to remove the ethanol solution to form a pre-treated tissue;
  • Pulverization pulverize the tissue to be pulverized obtained in step (3) and then sieve to form pulverized animal tissue of 0-300 ⁇ m;
  • the present embodiment provides a kind of preparation technology of high-yield collagen fiber, and described preparation technology comprises the following steps:
  • Pre-treatment soak the tissue pieces or tissue blocks obtained in step (1) in 1.0% sodium chloride solution, and mix for the first time at the ratio of adding 10ml sodium chloride solution per gram of tissue, Stir magnetically at 25-37°C for 24 hours, then wash with water to remove the sodium chloride solution, add 10ml of ethanol solution with a concentration of 75wt% to each gram of soaked tissue for the second mixing, stir magnetically at 25-37°C for 24h, wash with water Remove the ethanol solution to form a pre-treated tissue;
  • Pulverization pulverize the tissue to be pulverized obtained in step (3) and sieve to form pulverized animal tissue of 0-300 ⁇ m;
  • Alkali treatment In the solution containing 1.5M sodium hydroxide and 1.5M sodium sulfate, add 6wt% of the pulverized animal tissue obtained in step (4), and react at 25-37°C for 48h; after the reaction is completed Neutralize with 10M hydrochloric acid solution to pH 6.5, then neutralize with 1M sulfuric acid solution to pH 4.5, and then wash with water for 8 times to form collagen that maintains a triple helical structure.
  • the alkali treatment stage can effectively remove the terminal peptides and can obtain virus-inactivated tissues;
  • the steps of freeze-drying include in sequence:
  • Low-temperature freezing stage the temperature is -40°C, and the time is 350 minutes;
  • Vacuum drying stage temperature is -18°C, 720min, vacuum degree is 0.2bar;
  • the first drying stage the temperature is -12°C, 400min, the vacuum is 0.2bar,
  • the second drying stage the temperature is 14°C, 400min, and the vacuum is 0.2bar;
  • the third drying stage the temperature is 0°C, 90min, and the vacuum is 0.2bar;
  • the fourth drying stage the temperature is 25°C, 90min, and the vacuum is 0.2bar.
  • Example 1 The difference between this example and Example 1 is that the amount of ficin added is different, and the amount of added ficin in this example is 0.1 wt% of the pretreated tissue.
  • Example 1 The difference between this example and Example 1 is only that the amount of ficin added is different, and the amount of added ficin in this example is 2 wt% of the pretreated tissue.
  • This comparative example provides a kind of preparation technology of high-yield collagen fiber
  • the reference document of this comparative example pepsin extracts the research of pigskin collagen
  • described preparation process comprises the following steps:
  • Pretreatment and acid pickling Weigh the dry skin (about 5g at M 0 ), soak it in Tris-HCl buffer solution (mass ratio 1:20) with pH 7.4 for 2h, store at 4°C, and stir at intervals; after 2h Pour off the clear liquid, add a certain amount of 0.5M HAC solution to adjust the pH value of the solution, and let it soak for 2 hours;
  • step (3) Salting out: Centrifuge the solution obtained in step (2) (8000r/min, 15min), carefully absorb the supernatant, adjust the pH value to about 7.50 with sodium hydroxide solution, and use (NH 4 ) 2 SO 4 The final concentration is 1.5mol/L, add slowly, stir to dissolve, and let stand overnight.
  • step (4) Dialysis and drying: Pour the solution obtained in step (4) into a dialysis bag with a dialysis molecular mass of 8-10KDa, first dialyze with 0.04mol/L and 0.02mol /L Na2HPO3 solution for 2d, and then use Distilled water was dialyzed for 3 days, and the samples were freeze-dried and sealed.
  • Example 3 of the present invention The water consumption in the preparation process of Example 3 of the present invention was compared with the water consumption in the preparation process of Comparative Example 1, and the results are shown in Table 1.
  • Table 1 embodiment 3 water consumption compares with comparative example 1 water consumption
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to measure the molecular weight and miscellaneous protein content of the collagen obtained in step (5) of the three embodiments of the present invention, and the results are shown in FIG. 1 .
  • Fig. 1 is the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis method) diagram of the collagen prepared according to Examples 3-5 of the present invention.
  • Marker is a series of standard collagen samples with known relative molecular weights
  • sample a dissolve the collagen samples prepared in Examples 3-5 of the present invention with 3% acetic acid respectively, and the concentration of the solution is 1mg /mL, named as Line1 (embodiment 3), line3 (embodiment 4), line5 (embodiment 5) respectively; /mL, take 1 mg of the collagen samples prepared in Examples 3-5 of the present invention, respectively, and add them to 1 mL of collagenase aqueous solution to obtain a collagen sample solution with a concentration of 1 mg/mL, act in a water bath at 37°C for 4 hours, and name them as line2 (Example 3), line4 (Example 4), line6 (Example 5); sample c: ultrapure water dissolves collagenase, its concentration is identical with the concentration in sample b, and the concentration of collagenase aqueous solution is 0.05mg/mL
  • A is the sum of optical densities of all bands in sample a, %;
  • B is the sum of optical densities of all bands in sample b, %;
  • C is the sum of optical densities of all bands in sample c, %;
  • the impurity protein content in the collagen prepared by the present invention is 0.2%, and the purity of the collagen is as high as 99.8%.
  • the collagen of the present invention has high purity, low miscellaneous protein content, and miscellaneous protein ⁇ 0.25%. From the results of the SDS-PAGE figure, it can be seen that the collagen sample prepared by the present invention has obvious ⁇ component and ⁇ component And the ⁇ component, the ⁇ component is the ⁇ peptide chain, the ⁇ component is the dimer of the ⁇ peptide chain, and the ⁇ component is the trimer of the ⁇ peptide chain, which well maintains the original structure of the collagen.
  • Example 3 of the present invention The collagen sponge obtained in Example 3 of the present invention is crushed into different sizes and marked as sample 1, sample 2 and sample 3, and two kinds of acid-soluble collagen purchased from the market are respectively samples 4 and 5; respectively detect the bursting of the above samples Strength, tensile strength and degradation time.
  • Bursting strength Clamp the sample on the extensible diaphragm, apply liquid pressure under the diaphragm, and increase the volume of the liquid at a constant speed, so that the diaphragm and the sample expand until the sample ruptures to measure the bursting strength.
  • Tensile strength Take 5 samples with a width of 15mm and a spacing of 40mm, stretch at a speed of 10mm/min, record the maximum value at the time of breaking, and take the average value of the results;
  • Degradation time Weigh 1500mg of each sample, soak in 100ml 5U/mL collagenase PBS buffer solution, place the solution on a shaker at 37°C, and evaluate its degradation time;
  • the collagen sponge of the present invention has higher tensile strength, burst strength and longer degradation cycle.

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Abstract

一种高产率胶原海绵的制备工艺，选取富含胶原的动物组织，切成片或块，形成组织片或组织块；经前处理、酶反应、粉碎、碱处理、得到的保持三股螺旋结构的胶原；然后经冻干过程制成胶原海绵。有益效果：本方案在去除端肽、弹性蛋白、灭活病毒的前提下，具有更高的提取效率，杂蛋白去除彻底，所制得的胶原纯度较高。与酸溶胶原海绵相比，本方案制备的胶原海绵具有更高的抗拉强度、胀破强度和更长的降解周期。同时，工艺用水的使用量较少，反应装置的规模与其他酸或酶提取法相比较小，可大幅度节约生产场地。

Description

一种高产率胶原海绵的制备工艺 技术领域
本发明涉及一种胶原蛋白纤维，具体涉及一种用于医疗器械的高产率胶原海绵的制备工艺。
背景技术
胶原蛋白是细胞外基质主要成分之一，约占哺乳动物蛋白总质量的1/3，是构成皮肤、韧带、软骨、肌腱等结缔组织或器官的主要成分，胶原具有独特的理化性质和优良的生物相容性、可降解性、低免疫性等优点，使得其在生物体内容易被吸收、亲水性强、无毒安全性好，成为生物医用材料的选择之一，一般从富含胶原纤维的动物组织进行提取，如皮肤、肌腱、骨骼等。胶原的提取方法通常有碱法、盐法、酸法、酶法，依照其溶解性，可将胶原分为：胶原纤维及酸溶性胶原、水溶性胶原等。
中国专利文献CN101569765A涉及到一种保持胶原特有三螺旋结构的Ⅰ型医用胶原材料以及提取方法，应用于组织修复方面，同时该材料具有一定的抗拉力强度，减少组织粘连，有利于组织的修复与再生。其方案如下：
前处理：牛跟腱组织经冷冻切割成0.1-3mm的薄片，在搅拌作用下将其浸泡在0.1％磷酸二氢钠/氢氧化钠溶液中1-3小时，保持中性条件。
酶反应：将1:5(w/w)的无花果酶加入上述溶液中充分反应，搅拌4h，之后用1％的硝酸铵反应6h，清洗。
盐析：加入1M的氯化钠处理并控制温度，调节酸碱度，4℃条件下静置过夜，取出后用2M氢氧化钠溶液反应，后用50％硫酸中和缓冲，使溶液呈偏酸性，取出沉淀物。
清洗：将沉淀物水洗浸泡6h后去除水分，冷冻干燥的方法制得保持胶原特有三螺旋结构的Ⅰ型医用胶原材料。
冻干成膜：将上述得到的材料溶于0.01-0.5M的冰醋酸中，得到0.5％-2％ 的溶液，在15000-30000rad/min、0-15℃条件下于1-8h加入0.05％-60％的氨基葡聚糖，制成复合悬浮液，经冷冻干燥后，再经物理或化学方法加强胶原分子间的链连接，通过低温环氧乙烷消毒灭菌，制成孔隙在160-350μm之间具有三维孔隙结构的膜片。
然而，上述方法得到的悬浮液并不能以很好的方式将各组分分散均匀，抗拉强度低、降解速率快。
发明内容
为了解决现有技术中提取工序周期长，规模化生产占地面积大，以及可能掺入杂蛋白等问题，本发明提供了一种高产率胶原蛋白纤维的制备工艺，制备过程中并未破坏胶原结构，经过一系列条件处理，最终得到的是胶原纤维，采用胶原纤维制备的胶原海绵具有更高的抗拉强度、胀破强度和更长的降解周期。
本发明的目的是提供一种高产率胶原蛋白纤维的制备工艺。
根据本发明的具体实施方式的高产率胶原蛋白纤维的制备工艺，所述制备工艺包括以下步骤：
(1)切割：选取富含胶原的动物组织，切成片或块，形成组织片或组织块；
(2)前处理：将步骤(1)得到的所述组织片或组织块浸泡在氯化钠溶液中，氯化钠溶液的浓度为0.5-1.5wt％，磁力搅拌进行第一次混合，然后水洗去除氯化钠溶液；然后向浸泡后组织中加入乙醇溶液，所述乙醇溶液的浓度为60-90wt％，磁力搅拌进行第二次混合，然后水洗去除乙醇溶液，形成前处理组织；
(3)酶反应：在酸性或弱碱性溶液中，加入10-20wt％步骤(2)得到的所述前处理组织，再加入蛋白酶进行酶解，酶解完成后中止反应；然后水洗去除溶液，形成待粉碎组织；
(4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成粉碎后的动物组织；
(5)碱处理：在含有氢氧化钠和硫酸钠的溶液中，加入10-20wt％步骤(4)得到的所述粉碎后的动物组织进行反应；反应完成后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原；
(6)冻干成膜：在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.4-0.6wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.04-0.06wt％；将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
一般的牛皮肤、跟腱等富含胶原的动物组织结构较为致密，不易打碎，本发明中先将这些组织进行切割和前处理，可去除弹性蛋白以及可使富含胶原的组织结构松散，这样更容易打碎。
步骤(3)中，酶解可以采用无花果蛋白酶、胃蛋白酶或胰蛋白酶，经多次试验表明采用无花果蛋白酶对前处理组织进行酶解，酶解效果更好，得到的胶原纯度更高。
进一步的，步骤(3)中，所述蛋白酶为无花果蛋白酶，在pH为5.5-6.8的醋酸-醋酸钠缓冲溶液中，加入10-20wt％所述前处理组织，再加入所述前处理组织0.1-2wt％的无花果蛋白酶，在25-37℃磁力搅拌反应0.5-5h，反应完成后加入亚氯酸钠、硝酸铵、双氧水中的一种或几种组合，在25℃-37℃下反应0.5-2h，中止酶反应，水洗去除溶液，形成待粉碎组织。
进一步的，步骤(3)中，所述蛋白酶为胃蛋白酶，在pH为2.5-4.5的酸性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胃蛋白酶，在25-37℃下，反应2-12h，反应完成后加入0.5-10M氢氧化钠中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。优选的，先用浓的碱溶液(10M氢氧化钠)进行中和，快接近所需pH时，再用稀的碱溶液(0.5M氢氧化钠)进行中和至pH为5.0，减少水的用量。
进一步的，步骤(3)中，所述蛋白酶为胰蛋白酶，在pH为7.5-7.8的弱碱性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胰蛋白酶，在25-37℃下，反应2-12h，反应完成后加入1M盐酸溶液中和溶液 至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。
一般终止酶反应采用高温灭酶的方法，本发明中若采用高温会让胶原变性。采用pH值变化不足以终止无花果蛋白酶反应，因此，加入亚氯酸钠、硝酸铵、双氧水中的一种或几种组合终止酶反应效果更好。进一步的，步骤(3)中，若酶解组织中只加入亚氯酸钠，则所述亚氯酸钠的添加量为所述前处理组织的0.05-0.5wt％；若酶解组织中只加入硝酸铵，则所述硝酸铵的添加量为所述前处理组织的1-10wt％；若酶解组织中只加入双氧水，则所述双氧水的添加量为所述前处理组织的1-10wt％。酶解过程可有效去除弹性蛋白。同时，酶反应阶段的另两个替代方案也可有效去除弹性蛋白，不会对胶原纤维的结构发生破坏。
进一步的，步骤(1)中，所述动物富含胶原的组织为牛或猪的皮肤、跟腱。
优选的，将所述动物富含胶原的组织切成0.3-1.5mm厚的薄片或0.3-1.0mm的立方小块。
进一步的，步骤(2)中，所述组织片或组织块浸泡在0.5-1.5％的氯化钠溶液中，以每克组织中加入5-10ml的氯化钠溶液的比例进行第一次混合，在25-37℃下磁力搅拌12-36h，然后水洗去除氯化钠溶液；以每克浸泡后组织中加入5-10ml浓度为60-90wt％的乙醇溶液的比例进行第二次混合，在25-37℃下磁力搅拌12-36h，水洗去除乙醇溶液，形成前处理组织。
进一步的，步骤(4)中，所述粉碎后动物组织的粒径为0-300μm。
进一步的，步骤(5)中，在含有1.0-2.0M氢氧化钠和1.0-2.0M硫酸钠的混合溶液中，加入10-20wt％所述粉碎后动物组织，在25-37℃下反应36-72h；后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原蛋白。
碱处理阶段可以有效去除端肽并可得到病毒灭活的组织。混合溶液中氢氧化钠的浓度为1.0-2.0M，硫酸钠的浓度为1.0-2.0M，制备时先加硫酸钠，硫酸钠起到保护作用，没有硫酸钠溶液直接加碱溶液动物组织易变性。
进一步的，中和所用的酸为盐酸或硫酸；所用酸的浓度为0.5-10M。
进一步的，步骤(6)中，在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.5wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.05wt％；用均质机进行均质2h，将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
更进一步的，取步骤(5)制备的纯度高达99％的保持三股螺旋结构的胶原，并加入0.05％硫酸软骨素钠，用均质机在0.05M醋酸溶液中混合2h。将所得的溶液倒入医用不锈钢冷冻盘中，用低温冷冻-冷凝-升华-升温的冷冻干燥的方式制成胶原海绵。本发明中醋酸的作用：胶原在酸性条件下溶胀，才能在均质后均匀分散，而醋酸在后续冻干过程中可完全挥发。本发明中硫酸软骨素钠的作用：硫酸软骨素钠是细胞外基质的主要功能组分，其本身也具有细胞相互作用，与胶原蛋白共混发生共价键结合，增强诱导组织再生能力，同时改善产品力学及抗降解性能。
冷冻干燥的步骤依次包括：
低温冷冻阶段：温度为-40℃，时间350min；
真空干燥阶段：温度为-18℃，720min，真空度为0.2bar；
第一干燥阶段：温度为-12℃，400min，真空度为0.2bar；
第二干燥阶段：温度为14℃，400min，真空度为0.2bar；
第三干燥阶段：温度为0℃，90min，真空度为0.2bar；
第四干燥阶段：温度为25℃，90min，真空度为0.2bar。
与现有技术相比，本发明具有如下有益效果：
(1)本发明在去除端肽、弹性蛋白、灭活病毒的前提下，具有更高的提取效率，杂蛋白去除彻底，所制得的胶原蛋白纯度较高。
(2)同时本发明动物组织或胶原蛋白占溶液的重量比决定了工艺用水的使用量以及反应装置的规模，本发明所有步骤中最低质量比与其他酸/酶提取法相比提高了近10倍，可大幅度节约生产场地。
(3)本发明所制得的胶原蛋白纯度较高，杂蛋白含量较低(＜0.25％)。
与酸溶胶原海绵相比，本发明制备的胶原海绵具有更高的抗拉强度、胀破强度和更长的降解周期。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1显示根据本发明的具体实施例3的胶原的SDS-PAGE图谱；其中line1、line3、line5为酸溶胶原；line2、line4、line6为胶原酶酶解解胶原；line7为胶原蛋白酶水溶液。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
以下实施例中采用的无花果蛋白酶均为植物提取的蛋白酶，采购自MP Biomedicals LLC；酶活≥200Bapa U/g。
在一些较为具体的实施方案中，所述高产率胶原蛋白纤维的制备工艺包括以下步骤：
(1)切割：选取富含胶原的动物组织，切成片或块，形成组织片或组织块；
(2)前处理：将步骤(1)得到的所述组织片或组织块浸泡在氯化钠溶液中，氯化钠溶液的浓度为0.5-1.5wt％，磁力搅拌进行第一次混合，然后水洗去除氯化钠溶液，洗净形成浸泡后组织；向所述浸泡后组织中加入乙醇溶液，所述乙醇溶液的浓度为60-90wt％，磁力搅拌进行第二次混合，然后水洗去除乙醇溶液，形成前处理组织；
(3)酶反应：在酸性或弱碱性溶液中，加入10-20wt％步骤(2)得到的所述前处理组织，再加入蛋白酶进行酶解，酶解完成后中止反应；然后水洗去除溶液，形成待粉碎组织；
(4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成粉碎后动物组织；
(5)碱处理：在含有氢氧化钠和硫酸钠的溶液中，加入10-20wt％步骤(4)得到的所述粉碎后动物组织进行反应；反应完成后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原；
(6)冻干成膜：在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.4-0.6wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.04-0.06wt％；将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
进一步的，步骤(3)中，所述蛋白酶为无花果蛋白酶，在pH为5.5-6.8的醋酸-醋酸钠缓冲溶液中，加入10-20wt％所述前处理组织，再加入所述前处理组织0.1-2wt％的无花果蛋白酶，在25-37℃磁力搅拌反应0.5-5h，反应完成后加入亚氯酸钠、硝酸铵、双氧水中的一种或几种组合，在25℃-37℃下反应0.5-2h，中止酶反应，水洗去除溶液，形成待粉碎组织。
进一步的，步骤(3)中，所述蛋白酶为胃蛋白酶，在pH为2.5-4.5的酸性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胃蛋白酶，在25-37℃下，反应2-12h，反应完成后加入0.5-10M氢氧化钠中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。
进一步的，步骤(3)中，所述蛋白酶为胰蛋白酶，在pH为7.5-7.8的弱碱性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胰蛋白酶，在25-37℃下，反应2-12h，反应完成后加入0.5-10M盐酸中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。
进一步的，步骤(3)中，若酶解组织中只加入亚氯酸钠，则所述亚氯酸钠的添加量为所述前处理组织的0.05-0.5wt％；若酶解组织中只加入硝酸铵， 则所述硝酸铵的添加量为所述前处理组织的1-10wt％；若酶解组织中只加入双氧水，则所述双氧水的添加量为所述前处理组织的1-10wt％。
进一步的，步骤(1)中，所述动物富含胶原的组织为牛或猪的皮肤、跟腱；优选的，将所述动物富含胶原的组织切成0.3-1.5mm厚的薄片或0.3-1.0mm的立方小块。
进一步的，步骤(2)中，所述组织片或组织块浸泡在0.5-1.5％的氯化钠溶液中，以每克组织中加入5-10ml的氯化钠溶液的比例进行第一次混合，在25-37℃下磁力搅拌12-36h，然后水洗去除氯化钠溶液；以每克浸泡后组织中加入5-10ml浓度为60-90wt％的乙醇溶液的比例进行第二次混合，在25-37℃下磁力搅拌12-36h，水洗去除乙醇溶液，形成前处理组织。
进一步的，步骤(4)中，所述粉碎后动物组织的粒径为0-300μm。
进一步的，步骤(5)中，在含有1.0-2.0M氢氧化钠和1.0-2.0M硫酸钠的混合溶液中，加入10-20wt％所述粉碎后动物组织，在25-37℃下反应36-72h；后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原。
进一步的，中和所用的酸为盐酸或硫酸；所用酸的浓度为0.5-10M。
进一步的，步骤(6)中，向所述保持三股螺旋结构的胶原中加入0.05wt％的硫酸软骨素钠，混合均匀后加入到0.05M醋酸溶液中用均质机进行均质2h，将均质后所得的溶液进行冷冻干燥，制成胶原蛋白海绵。
以下通过实施例并结合附图进一步详细说明本发明的技术方案。然而，所选的实施例仅用于说明本发明，而不限制本发明的范围。
实施例1
本实施例提供了一种高产率胶原蛋白纤维的制备工艺，所述制备工艺包括以下步骤：
(1)切割：选取猪皮，切成薄片，形成组织片；
(2)前处理：将步骤(1)得到的所述组织片浸泡在浓度为1.5％的氯化钠溶液中，磁力搅拌进行第一次混合，然后水洗去除氯化钠溶液，向浸泡后组织 中加入乙醇溶液，所述乙醇溶液的浓度为90wt％，磁力搅拌进行第二次混合，然后水洗去除乙醇溶液，形成前处理组织；
(3)酶反应：在pH为7.6的弱碱性溶液中，加入15wt％前处理组织，再加入所述前处理组织10wt％的胰蛋白酶，在25℃下，反应10h，反应完成后加入1M盐酸溶液中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织；
(4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成粉碎后动物组织；
(5)碱处理：在含有氢氧化钠和硫酸钠的溶液中，加入10wt％步骤(4)得到的所述粉碎后动物组织进行反应；反应完成后用酸中和至pH为4.0，再用清水进行清洗5次，形成保持三股螺旋结构的胶原；
(6)冻干成膜：在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.6wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.04wt％；将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
实施例2
本实施例提供了一种高产率胶原蛋白纤维，制备工艺包括以下步骤：
(1)切割：选取牛皮，切成长、宽均为0.3-1.5mm的薄片，形成组织块；
(2)前处理：将步骤(1)得到的所述组织片浸泡在浓度为1.5％的氯化钠溶液中，以每克组织中加入10ml的氯化钠溶液的比例进行第一次混合，在25-37℃下磁力搅拌36h，然后水洗去除氯化钠溶液；以每克浸泡后组织中加入5ml浓度为60wt％的乙醇溶液的比例进行第二次混合，在25-37℃下磁力搅拌12h，水洗去除乙醇溶液，形成前处理组织；
(3)酶反应：在pH为2.5的酸性溶液中，加入15wt％前处理组织，再加入所述前处理组织10wt％的胃蛋白酶，在28℃下，反应10h，反应完成后加入1M氢氧化钠中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织；
(4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成0-300μm粉碎后的动物组织；
(5)碱处理：在含有2.0M氢氧化钠和1.0M硫酸钠的溶液中，加入10wt％步骤(4)得到的所述粉碎后动物组织，在25-37℃下反应72h；反应完成后用浓度为10M的盐酸溶液中和至pH为7.0，之后用0.5M的稀盐酸中和至pH为5.0，再用清水进行清洗10次，形成保持三股螺旋结构的胶原，碱处理阶段可以有效去除端肽并可得到病毒灭活的组织；
(6)冻干成膜：在100ml 0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.4wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.06wt％；用均质机进行均质2h，将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
实施例3
本实施例提供了一种高产率胶原蛋白纤维的制备工艺，所述制备工艺包括以下步骤：
(1)切割：选取牛跟腱，切成0.3-1.0mm的立方小块，形成组织块；
(2)前处理：将步骤(1)得到的所述组织片或组织块浸泡在1.0％的氯化钠溶液中，以每克组织中加入10ml氯化钠溶液的比例进行第一次混合，在25-37℃下磁力搅拌24h，然后水洗去除氯化钠溶液，每克浸泡后组织中加入10ml浓度为75wt％的乙醇溶液进行第二次混合，在25-37℃下磁力搅拌24h，水洗去除乙醇溶液，形成前处理组织；
(3)酶反应：在pH为6.0的醋酸-醋酸钠缓冲溶液中，加入15wt％步骤(2)得到的所述前处理组织，再加入1.0wt％无花果蛋白酶进行酶解，在25-37℃磁力搅拌反应3h，清水洗净，形成酶解组织；然后加入双氧水，双氧水的添加量为所述前处理组织的5％，在25℃-37℃下反应1h，中止反应，水洗去除溶液，形成待粉碎组织；
(4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成 0-300μm的粉碎后动物组织；
(5)碱处理：在含有1.5M氢氧化钠和1.5M硫酸钠的溶液中，加入6wt％步骤(4)得到的所述粉碎后动物组织，在25-37℃下反应48h；反应完成后用浓度为10M的盐酸溶液中和至pH为6.5，之后用1M的硫酸溶液中和至pH为4.5，再用清水进行清洗8次，形成保持三股螺旋结构的胶原，碱处理阶段可以有效去除端肽并可得到病毒灭活的组织；
(6)冻干成膜：在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.5wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.05wt％；用均质机进行均质2h，将均质后所得的溶液倒入医用不锈钢冷冻盘中，用低温冷冻-冷凝-升华-升温的冷冻干燥的方式制成胶原海绵。
冷冻干燥的步骤依次包括：
低温冷冻阶段：温度为-40℃，时间350min；
真空干燥阶段：温度为-18℃，720min，真空度为0.2bar；
第一干燥阶段：温度为-12℃，400min，真空度为0.2bar，
第二干燥阶段：温度为14℃，400min，真空度为0.2bar；
第三干燥阶段：温度为0℃，90min，真空度为0.2bar；
第四干燥阶段：温度为25℃，90min，真空度为0.2bar。
实施例4
本实施例与实施例1的区别仅在于：无花果蛋白酶的添加量不同，本实施例无花果蛋白酶的添加量为所述前处理组织的0.1wt％。
实施例5
本实施例与实施例1的区别仅在于：无花果蛋白酶的添加量不同，本实施例无花果蛋白酶的添加量为所述前处理组织的2wt％。
对比例1
本对比例提供了一种高产率胶原蛋白纤维的制备工艺，本对比例的参考文献：胃蛋白酶提取猪皮胶原的研究，所述制备工艺包括以下步骤：
(1)预处理与浸酸:称取干燥皮块(M 0约5g)，用pH7.4的Tris－HCl缓冲液(质量比1:20)浸泡2h，4℃保存，间隔搅拌；2h后倒去清液，加入一定量的0.5M的HAC溶液调节溶液pH值，静置浸泡2h；
(2)酶解:将步骤(1)得到的溶液准确加入4％的胃蛋白酶(1∶3000)，连续搅拌26h。
(3)盐析:将步骤(2)得到的溶液离心分离(8000r/min，15min)，小心吸取上清液，用氢氧化钠溶液调pH值为7.50左右，以(NH 4) 2SO 4最终浓度为1.5mol/L，缓慢加入，搅拌使之溶解，静置过夜。
(4)酸解:将步骤(3)在步骤(2)离心的条件下进行离心，取沉淀，加入0.5M HAC溶液，在4℃下搅拌30min，得到胶原蛋白溶液母液。
(5)透析与干燥:将步骤(4)得到的溶液倒入透析分子质量为8～10KDa的透析袋中，先用0.04mol/L和0.02mol/L Na 2HPO 3溶液透析2d，然后用蒸馏水透析3d，将试样冷冻干燥，后密封。
将本发明实施例3的制备过程中的用水量和对比例1制备过程中的用水量进行比较，结果如表1。
表1实施例3用水量与对比例1用水量比较
Figure PCTCN2022134658-appb-000001
从表1结果中得知，实施例3每步用水量相较于对比例每步用水量都少，同时，实施例3所有步骤中最低质量比与对比例1相比高了近10倍，大幅度节约生产场地和工艺用水量，说明本发明的高产率胶原蛋白纤维的制备工艺更加节能，生产成本更低。
性能测试试验
采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对本发明3个实施例步骤(5)得到的胶原进行分子量及杂蛋白含量的测定，结果如图1所示。
胶原相对分子质量的测定方法有很多种，但应用较多的是聚丙烯酰胺凝胶电泳法，同时用胶原蛋白酶可以特异性地水解天然胶原的三股螺旋结构，检测胶原蛋白样品中所含杂蛋白含量。通过考马斯亮蓝对牛血清白蛋白(BSA)染色极限的确定，检测胶原蛋白样品中所含杂蛋白总量。具体可参考YY0954。图1是根据本发明的实施例3-5所制备得到的胶原的SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法)图。
样品中杂蛋白分析：Marker为一系列的已知相对分子质量的标准胶原蛋白 样品，样品a:分别用3％的乙酸溶解本发明实施例3-5所制备的胶原样品，溶液的浓度为1mg/mL，分别命名为Line1(实施例3)、line3(实施例4)、line5(实施例5)；样品b：超纯水溶解胶原蛋白酶得到胶原蛋白酶浓度水溶液，胶原蛋白酶水溶液的浓度为0.05mg/mL，分别取本发明实施例3-5所制备的胶原样品1mg，分别加入到1mL胶原蛋白酶水溶液，得到胶原样品溶液的浓度均为1mg/mL，于37℃水浴作用4h，分别命名为line2(实施例3)、line4(实施例4)、line6(实施例5)；样品c：超纯水溶解胶原蛋白酶，其浓度与样品b中的浓度相同，胶原蛋白酶水溶液的浓度为0.05mg/mL，命名为line7。
纯度计算：当B-C≠0时，样品中胶原蛋白纯度(％)＝A-(B-C)；当B-C＝0时，样品中胶原蛋白纯度(％)＝(10000-BSA极限值)/100，(经检测，本预制胶的BSA极限值为20)。
A为样品a所有条带光密度之和，％；
B为样品b所有条带光密度之和，％；
C为样品c所有条带光密度之和，％；
通过计算可得知，本发明制得的胶原蛋白中的杂蛋白含量是0.2％，胶原蛋白的纯度高达99.8％。
结果表明，本发明的胶原的纯度较高，杂蛋白含量较低，杂蛋白＜0.25％，从SDS-PAGE图的结果可知：本发明制备得到的胶原样品具有明显的α组分、β组分及γ组分，α组分即为α肽链,β组分为α肽链的二聚体，γ组分为α肽链的三聚体，很好的保持了胶原原有的结构。
2、将本发明实施例3得到的胶原海绵粉碎成不同尺寸标记为样品1、样品2和样品3，从市场购买的两种酸溶胶原分别为样品4、5；分别检测上述样品的胀破强度、抗拉强度及降解时间。
(1)测试方法
胀破强度：将试样夹在可延伸的膜片上，在膜片下面施加液体压力，并以恒定速度增加液体体积，使膜片和试样膨胀直至试样破裂即测得胀破强度。
抗拉强度：取样品5条，宽度为15mm，间距40mm，以10mm/min速度 进行拉伸，记录断裂时的最大值，结果取平均值；
降解时间：称重各个样品1500mg，浸泡在100ml 5U/mL胶原酶的PBS缓冲溶液中，溶液放在37℃的摇床上，评估其降解时间；
(2)试验结果如下表2：
表2胶原样品的胀破强度、抗拉强度和降解时间的比较
Figure PCTCN2022134658-appb-000002
从表2可以看出，与市场购买的两种酸溶胶原海绵相比，本发明的胶原海绵具有更高的抗拉强度、涨破强度和更长的降解周期。纤维尺寸＞150μm，抗拉强度为8.2N，纤维尺寸100μm-150μm，抗拉强度为10.3N，纤维尺寸＜100μm，抗拉强度为9.7N。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种高产率胶原海绵的制备工艺，其特征在于，所述制备工艺包括以下步骤：
    (1)切割：选取富含胶原的动物组织，切成片或块，形成组织片或组织块；所述富含胶原的动物组织为牛或猪的皮肤或跟腱；
    (2)前处理：将步骤(1)得到的所述组织片或组织块浸泡在氯化钠溶液中，氯化钠溶液的浓度为0.5-1.5wt％，磁力搅拌进行第一次混合，然后水洗去除氯化钠溶液；然后向浸泡后组织中加入乙醇溶液，所述乙醇溶液的浓度为60-90wt％，磁力搅拌进行第二次混合，然后水洗去除乙醇溶液，形成前处理组织；
    (3)酶反应：在酸性或弱碱性溶液中，加入10-20wt％步骤(2)得到的所述前处理组织，再加入蛋白酶进行酶解，酶解完成后中止反应；然后水洗去除溶液，形成待粉碎组织；
    (4)粉碎：将步骤(3)得到的所述待粉碎组织进行粉碎后过筛，形成粉碎后动物组织；
    (5)碱处理：在含有氢氧化钠和硫酸钠的溶液中，加入10-20wt％步骤(4)得到的所述粉碎后动物组织进行反应；反应完成后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原；
    (6)冻干成膜：在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.4-0.6wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.04-0.06wt％；将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
  2. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(3)中，所述蛋白酶为无花果蛋白酶，在pH为5.5-6.8的醋酸-醋酸钠缓冲溶液中，加入10-20wt％所述前处理组织，再加入所述前处理组织0.1-2wt％的 无花果蛋白酶，在25-37℃磁力搅拌反应0.5-5h，反应完成后加入亚氯酸钠、硝酸铵、双氧水中的一种或几种组合，在25℃-37℃下反应0.5-2h，中止酶反应，水洗去除溶液，形成待粉碎组织。
  3. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(3)中，所述蛋白酶为胃蛋白酶，在pH为2.5-4.5的酸性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胃蛋白酶，在25-37℃下，反应2-12h，反应完成后加入0.5-10M氢氧化钠中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。
  4. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(3)中，所述蛋白酶为胰蛋白酶，在pH为7.5-7.8的弱碱性溶液中，加入10-20wt％前处理组织，再加入所述前处理组织1.0-15wt％的胰蛋白酶，在25-37℃下，反应2-12h，反应完成后加入0.5-10M盐酸溶液中和溶液至pH为5.0，中止酶反应，水洗去除溶液，形成待粉碎组织。
  5. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(2)中，所述组织片或组织块浸泡在0.5-1.5％的氯化钠溶液中，以每克组织中加入5-10ml的氯化钠溶液的比例进行第一次混合，在25-37℃下磁力搅拌12-36h，然后水洗去除氯化钠溶液，以每克浸泡后组织中加入5-10ml浓度为60-90wt％的乙醇溶液的比例进行第二次混合，在25-37℃下磁力搅拌12-36h，水洗去除乙醇溶液，形成前处理组织。
  6. 根据权利要求2所述的高产率胶原海绵的制备工艺，其特征在于，步骤(3)中，若酶解组织中只加入亚氯酸钠，则所述亚氯酸钠的添加量为所述前处理组织的0.05-0.5wt％；若酶解组织中只加入硝酸铵，则所述硝酸铵的添加量为所述前处理组织的1-10wt％；若酶解组织中只加入双氧水，则所述双氧水的添加量为所述前处理组织的1-10wt％。
  7. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(4)中，所述粉碎后动物组织的粒径为0-300μm。
  8. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(5)中，在含有1.0-2.0M氢氧化钠和1.0-2.0M硫酸钠的溶液中，加入10-20wt％所述粉碎后动物组织，在25-37℃下反应36-72h；后用酸中和至pH为4.0-5.0，再用清水进行清洗，形成保持三股螺旋结构的胶原。
  9. 根据权利要求1所述的高产率胶原海绵的制备工艺，其特征在于，步骤(6)中，在0.05M的醋酸溶液中，加入步骤(5)得到的所述保持三股螺旋结构的胶原和硫酸软骨素钠进行均质，所述保持三股螺旋结构的胶原的添加量为所述醋酸溶液0.5wt％，所述硫酸软骨素钠的添加量为所述醋酸溶液0.05wt％；用均质机进行均质2h，将均质后所得的溶液进行冷冻干燥，制成胶原海绵。
  10. 一种根据权利要求1-9任一所述的高产率胶原海绵的制备工艺制成的高产率胶原海绵。
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