WO2021135564A1 - 大网膜脱细胞基质材料的制备方法及软骨组织的构建方法 - Google Patents

大网膜脱细胞基质材料的制备方法及软骨组织的构建方法 Download PDF

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WO2021135564A1
WO2021135564A1 PCT/CN2020/123742 CN2020123742W WO2021135564A1 WO 2021135564 A1 WO2021135564 A1 WO 2021135564A1 CN 2020123742 W CN2020123742 W CN 2020123742W WO 2021135564 A1 WO2021135564 A1 WO 2021135564A1
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omentum
matrix material
acellular matrix
preparation
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万绵水
林创鑫
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广东泓志生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/3654—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C12N2533/92—Amnion; Decellularised dermis or mucosa

Definitions

  • the present invention relates to the technical field of tissue engineering, in particular to a method for preparing omentum acellular matrix material and a method for constructing cartilage tissue.
  • the commonly used repair methods still have certain limitations.
  • the traditional cartilage injury treatment methods include arthroplasty, drilling, microfracture and arthroscopic lavage. However, none of these methods can restore the damaged cartilage and subchondral bone to their original normal tissue structure.
  • Omentum is a type of peritoneum.
  • the peritoneum is a layer of mucosa in the abdominal cavity of higher vertebrates. It is mainly composed of mesothelial cells and is a membranous tissue formed by the support of connective tissue.
  • the peritoneum covers most of the organs in the abdominal cavity and can secrete mucus to moisten the surface of the organs and reduce the friction between the organs.
  • the peritoneum migrates from the parietal layer to the visceral layer, or from one organ to another, forming a double-layer structure. There are often blood vessels, nerves and lymphatic vessels running between the two layers of peritoneum.
  • the greater omentum is the peritoneum that connects the greater curvature of the stomach to the transverse colon.
  • the peritoneum including the anterior and posterior walls of the stomach, heals at the greater curvature to form the first two layers of the greater omentum, extending down to slightly below the umbilical plane. Then fold back upwards and coat the transverse colon to form the latter two layers of the greater omentum.
  • Omentum is a very thin tissue, rich in elasticity and flexibility, and has a highly vascularized structure. These characteristics make it an excellent natural biomaterial in the fields of tissue engineering and regenerative medicine.
  • the omentum can be used as a "natural bioreactor" for in vitro transplantation constructs, promote the formation of vascularization of the constructs, and enhance the function of the transplanted constructs. It has been successfully applied to myocardial tissue engineering and nerve tissue. Engineering and other fields. However, there is no research report on the application of omentum acellular matrix to cartilage tissue engineering.
  • the main purpose of the present invention is to provide a method for preparing acellular matrix material of the greater omentum and a method for constructing cartilage tissue to solve the above-mentioned problems of the existing acellular matrix material of the greater omentum.
  • the first aspect of the present invention provides a method for preparing acellular matrix material of the omentum, the preparation method comprising the steps:
  • the greater omentum is subjected to a freeze-thaw cycle treatment.
  • icing is formed and the concentration of the salt solution for the freeze-thaw cycle changes (the freezing process of the freeze-thaw cycle will cause the unfrozen water as the temperature drops.
  • the solubility of salt changes, and the salt concentration gradually rises, resulting in a concentration difference between the inside and outside of the cell membrane, causing the cells to rupture, thereby improving the cell lysis effect, and obtaining a larger total surface area suitable for tissue regeneration of cartilage tissue.
  • the pre-treatment process Before the degreasing treatment and the detissue cell treatment, the pre-treatment process must be carried out, that is, the carboxyl groups in the lysine and arginine residues are cut off by trypsin in a liquid environment with a pH value of 7.8 to 8.2, so as to break the cells.
  • many proteases will be released from the cells into the treatment solution, which will affect the other protein structures of the omentum tissue (the structure of the matrix material) Cause damage.
  • phenylmethylsulfonyl fluoride is added to the solution in the pre-treatment process, and phenylmethylsulfonyl fluoride is used as a protease inhibitor to prevent the aforementioned destruction process, thereby ensuring that the three-dimensional space of the resulting matrix material is more stable
  • the three-dimensional space of the matrix material generated by this method can promote the proliferation and migration of chondrocytes, and improve the stability of chondrocyte expression according to its original phenotype.
  • Pre-treatment and then decellularization treatment can improve the surface roughness of matrix particles, which is more conducive to the adhesion of cartilage cells and the regeneration of cartilage tissue.
  • Omentum can be selected from mammals such as pigs, cattle, sheep, etc., which can be purchased from slaughterhouses and other places.
  • physiological saline is used to clean the omentum.
  • Physiological saline has a certain bactericidal and sterilization effect, which can make bacteria produce a difference in intracellular and extracellular concentration, which can easily cause the effect of plasmolysis in bacterial cells and inhibit the reproduction of bacteria.
  • physiological saline has no harm to physiological cells to ensure large The integrity of the omentum.
  • the cleaning times can be 2-3 times.
  • the cleaned omentum is added to a buffer solution containing tris and phenylmethylsulfonyl fluoride with a pH value of 7.8 to 8.2 to perform the freezing and thawing cycle .
  • the use of the above buffer can improve the cell lysis effect
  • the addition of phenylmethylsulfonyl fluoride can provide a stable environment for the tissue, and prevent the protease released from the cell from affecting the other protein structures of the omentum tissue (constitute the matrix).
  • the structure of the material causes damage, thereby ensuring that the three-dimensional space of the generated matrix material is more stable.
  • the molar concentration of the tris is 1 mmol/L to 20 mmol/L, for example, it can be 1 mmol/L, 2 mmol/L, 3 mmol/L, 4 mmol/L, 5 mmol/L.
  • the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%, for example The mass percentage is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, more preferably 0.8% to 1%, and still more preferably 1%.
  • the buffer solution also contains ethylenediaminetetraacetic acid, and the molar concentration of the ethylenediaminetetraacetic acid is 1mmol/L to 10mmol/L, for example, 1mmol/L, 2mmol/L, 3mmol/L, 4mmol /L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, more preferably 8mmol/L to 10mmol/L, still more preferably 10mmol/L.
  • the molar concentration of the ethylenediaminetetraacetic acid is 1mmol/L to 10mmol/L, for example, 1mmol/L, 2mmol/L, 3mmol/L, 4mmol /L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, more preferably 8mmol/L to 10mmol/L, still more preferably
  • ethylenediaminetetraacetic acid to adjust the pH value of the buffer solution so that the pH value is controlled within the range of 7.8 to 8.2.
  • the use of ethylenediaminetetraacetic acid for pH adjustment can avoid the introduction of new The substance causes damage to other protein structures of the omentum tissue (the structure of the matrix material), thereby further ensuring that the three-dimensional space of the generated matrix material is more stable.
  • one freeze-thaw cycle in the freeze-thaw cycle includes: firstly perform a freezing treatment at a temperature of -80°C to -20°C for 1 to 8 hours, and the temperature of the freezing treatment may be, for example, -80°C or -75°C.
  • the length of freezing treatment such as It can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours; then conduct the melting treatment at a temperature of 30°C to 40°C for 0.5 to 1 hour.
  • the temperature of the melting treatment is for example It can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, and the duration of the melting treatment can be, for example, 0.5 hour, 0.6 hour, 0.7 hour , 0.8 hours, 0.9 hours, 1 hour.
  • the number of freeze-thaw cycles may be 2-3 times, more preferably 3 times.
  • the freeze-thaw cycle is an existing processing technology, due to the very demanding requirements of the omentum acellular matrix material, too high a melting temperature will lead to protein denaturation, and too low a melting temperature will lead to incomplete thawing, due to benzyl
  • the half-life of the protease inhibitor of sulfonyl fluoride is too short. If the thawing time is too long, it will cause the intracellular protease after cell lysis to enzymolyze the extracellular matrix. Therefore, the existing preparation process of omentum acellular matrix material This technology is not used in this application. In this application, the temperature and duration are limited, so that the freeze-thaw cycle can well rupture the cells without causing damage to the extracellular matrix.
  • the mass percentage of the trypsin is 0.1% to 0.5%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, more preferably 0.25%
  • the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, more preferably 0.8% to 1%, still more preferably 1%;
  • the mass percentage of the trypsin is 0.1% to 0.5%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, It is further preferably 0.25%, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, more preferably 0.8% to 1%, still more preferably 1%.
  • ethylenediaminetetraacetic acid is added to the solutions in the step S300 and the step S500, and the mass percentage of the ethylenediaminetetraacetic acid is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03 %, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, more preferably 0.8% to 0.1%, still more preferably 0.1%.
  • step S300 and step S500 ethylenediaminetetraacetic acid is used to adjust the pH value of the buffer solution so that the pH value is controlled within the range of 7.8 to 8.2.
  • ethylenediaminetetraacetic acid has an inhibitory effect, it is used for pH The value adjustment can avoid the damage to other protein structures (structures of the matrix material) of the omentum tissue due to the introduction of new substances, thereby further ensuring that the three-dimensional space of the generated matrix material is more stable.
  • the first predetermined time length is 6 to 12 hours, for example, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours; further preferably, in step S300, the first intermediate The substance is added to a solution containing trypsin and phenylmethylsulfonyl fluoride with a pH value of 7.8 to 8.2, shaking and continuing for the first predetermined time.
  • the second predetermined time length is 3 to 6 hours, for example, 3 hours, 4 hours, 5 hours, and 6 hours.
  • the third intermediate is added to a solution containing trypsin and phenylmethylsulfonyl fluoride with a pH value of 7.8 to 8.2, shaking and continuing for a second predetermined period of time.
  • step S400 one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol, and methanol is used to degrease the second intermediate. It is further preferred to use isopropanol to degrease the second intermediate. Compared with methanol, chloroform, and ether, isopropanol has a high boiling point, no pungent odor, is not easy to volatilize, and can reduce environmental pollution.
  • the second intermediate is put into one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol, and methanol, and under constant temperature conditions (30°C to 40°C, For example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C) shaking for 40 to 50 hours, such as 40 hours, 41 hours, 42 hours , 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, more preferably 48 hours.
  • a phosphate buffered saline solution is used for cleaning, and the number of times of cleaning is 2-3 times, and the pH value of the phosphate buffered saline solution is preferably 7.8 to 8.2, for example, 7.8, 7.9, 8, 8.1, 8.2 , More preferably 8.
  • a solution containing magnesium salt, deoxyribonuclease, ribonuclease, and lipase is used to deorganize the fourth intermediate.
  • the fourth intermediate is placed in a solution containing magnesium salt, deoxyribonuclease, ribonuclease and lipase under constant temperature conditions (30°C to 40°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C) for 10 to 32 hours, such as 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, more preferably 14 to 18 hours, still more preferably 16 hours.
  • step S600 a solution containing deoxyribonuclease, ribonuclease and lipase is used to deorganize the fourth intermediate.
  • the pretreatment process of step S500 makes the cells discrete and can effectively remove all cellular components.
  • the divalent magnesium ions in the salt have the effect of inhibiting trypsin, so that the deorganized cell treatment will not damage the matrix material and ensure the integrity of the matrix material.
  • the magnesium salt is one or a mixture of magnesium chloride and magnesium sulfate.
  • the molar concentration of the magnesium salt is 0.005mmol/L to 0.05mmol/L, for example 0.005mmol/L, 0.01mmol/L, 0.015mmol/L, 0.02mmol/L, 0.025mmol/L, 0.03mmol /L, 0.035mmol/L, 0.04mmol/L, 0.045mmol/L, 0.05mmol/L
  • the deoxyribonuclease content is 5KU/L to 50KU/L, for example, 5KU/L, 10KU/L, 15KU/L, 20KU/L, 25KU/L, 30KU/L, 35KU/L, 40KU/L, 45KU/L, 50KU/L, more preferably 10KU/L to 20KU/L, still more preferably 15KU/L
  • the enzyme type of deoxyribonuclease is Type II, Type II-S, or Type IV, and the content of the ribonuclease is 5 mg/L to 20 mg/L,
  • the phosphate buffered saline solution is used for cleaning first, and the pH value of the phosphate buffered saline solution is preferably 7.8 to 8.2, for example, 7.8, 7.9, 8, 8.1, 8.2, and more preferably 8.
  • the number of times can be 2-3 times, and then ethanol is used for cleaning, the ethanol concentration is preferably 50% to 70%, and the number of cleaning times can be 2-3 times.
  • freeze-drying is used for drying
  • irradiation sterilization is used for sterilization.
  • the obtained fourth intermediate is washed with a phosphate buffered saline solution with a pH value of 7.8 to 8.2 before entering the step S600.
  • the method of the present application can completely retain the three-dimensional structure of the extracellular matrix and some growth factors that play an important role in cell differentiation (such as transforming growth factor (TGF- ⁇ ), basic fibroblast growth factor (bFGF) ) And other growth factors), the three-dimensional space composed of the omentum acellular matrix material prepared by this method can promote the proliferation and migration of chondrocytes, improve the stability of chondrocyte expression according to their original phenotype, and promote the secretion of cell matrix. Increase the sturdiness of the bracket.
  • the omentum acellular matrix material prepared in the present application also has good biocompatibility, cell adsorption and hydrophilicity, and has strong degradation performance and host integration ability.
  • the second aspect of the present invention provides a method for constructing cartilage tissue, which includes the steps:
  • this step can amplify the number of cartilage cells
  • step S20 Take a cell suspension from the culture solution obtained in step S10, and inoculate it on the omentum acellular matrix material prepared by the above-mentioned preparation method;
  • the construction method provided in this application adsorbs seed cells in a scaffold carrier composed of a large omentum decellularized matrix material that has good biocompatibility and can be gradually absorbed by the human body.
  • the carrier provides a three-dimensional space for cells to survive, which is beneficial to Cells obtain sufficient nutrients, grow, and expand to obtain constructed cartilage tissue.
  • the constructed cartilage tissue forms an integral graft with the scaffold carrier. The graft is implanted into the tissue lesions in the body, and the cells are planted.
  • the seed cells used for cartilage tissue construction can be purchased in the cell bank, or cartilage tissue of patients or volunteers can be taken.
  • cartilage tissue of the patient it is preferable to take cartilage tissue rich in cartilage cells in the non-weight-bearing area.
  • the cartilage tissue in the non-weight-bearing area can be taken under aseptic conditions, washed 2 to 3 times in a culture bottle filled with nutrient solution, and then cut into small pieces under aseptic conditions Block and blot dry with sterile filter paper, and put it into a culture bottle filled with nutrient solution.
  • the nutrient solution is a DMEM/F12 culture solution containing fetal bovine serum
  • DMEM dulbecco's modified eagle medium
  • F12 Ham's F 12
  • Nutrient medium is an animal cell culture medium.
  • DMEM/F12 culture medium is obtained by combining DMEM and F12 at a ratio of 1:1.
  • the third predetermined period of time is preferably 6-8 weeks.
  • the culture solution includes DMEM culture solution. Because DMEM contains a variety of amino acids and glucose, it greatly improves the adhesion rate of cells.
  • the DMEM culture solution is supplemented with fetal bovine serum, penicillin, streptomycin, amphotericin, glutamine, vitamin additives and vitamin C.
  • the mass percentage of fetal bovine serum is 5% to 15%, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, and further Preferably it is 10%
  • the content of penicillin is 800IU/ml to 1200IU/ml, such as 800IU/ml, 900IU/ml, 1000IU/ml, 1100IU/ml, 1200IU/ml, more preferably 1000IU/ml
  • the content of streptomycin 0.8mg/ml to 1.2mg/ml, for example 0.8mg/ml, 0.9mg/ml, 1mg/ml, 1.1mg/ml, 1.2mg/ml, more preferably 1mg/ml
  • the content of amphotericin is 2
  • fetal bovine serum provides necessary nutrients for cells, performs acid-base balance, detoxification, and inhibits the activity of certain enzyme preparations; penicillin, streptomycin, and amphotericin are mainly used to eliminate pollution.
  • microbial pollution and the mixing of other cell lines. Both types of pollution are very common. We must pay attention to possible pollution and the impact of pollution on the results. Therefore, antibiotics and antifungal agents are used to prevent pollution; It is important during cell culture. After removing the amino group, L-glutamine can be used as an energy source for cultured cells, participating in protein synthesis and nucleic acid metabolism; vitamin additives provide nutrition; vitamin C plays a role in antioxidant protection for cells.
  • the fourth predetermined period of time is preferably 3-5 days.
  • the liquid level of the culture solution is higher than the surface of the omentum acellular matrix material, and the height difference is less than 1 mm.
  • the culture temperature in step S30 is between 35°C and 38°C, preferably 37°C, and the culture solution is preferably replaced once a day.
  • the present invention also provides a method for constructing cartilage tissue, which includes the steps:
  • step S09 taking a cell suspension from the culture solution obtained in step S08, and seeding it on the omentum acellular matrix material obtained in step S07;
  • the prepared omentum acellular matrix material has a large total surface area and a very stable three-dimensional space, which is very suitable for the subsequent regeneration and culture of cartilage tissue, promotes the proliferation and migration of chondrocytes, and improves the expression of chondrocytes according to their original phenotype.
  • the stability is very suitable for the subsequent regeneration and culture of cartilage tissue, promotes the proliferation and migration of chondrocytes, and improves the expression of chondrocytes according to their original phenotype.
  • physiological saline is used to clean the omentum.
  • the cleaned omentum is added to a buffer solution containing tris and phenylmethylsulfonyl fluoride with a pH value of 7.8 to 8.2 to perform the freezing and thawing cycle .
  • the molar concentration of the tris is 1 mmol/L to 20 mmol/L, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%.
  • the buffer solution also contains ethylenediaminetetraacetic acid, and the molar concentration of the ethylenediaminetetraacetic acid is 1 mmol/L to 10 mmol/L.
  • one freeze-thaw cycle in the freeze-thaw cycle includes: firstly perform a freezing treatment at a temperature of -80°C to -20°C for 1 to 8 hours, and then perform a freezing treatment at a temperature of 30°C to 40°C for 0.5 to 1 hour. Hours of melting treatment.
  • the mass percentage of the trypsin is 0.1% to 0.5%, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%;
  • the mass percentage of the trypsin is 0.1% to 0.5%, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%.
  • ethylenediaminetetraacetic acid is added to the solutions in step S03 and step S05, and the mass percentage of ethylenediaminetetraacetic acid is 0.01% to 0.1%.
  • one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol, and methanol is used to degrease the second intermediate.
  • a phosphate buffered saline solution is used for cleaning.
  • a solution containing magnesium salt, deoxyribonuclease, ribonuclease, and lipase is used to deorganize the fourth intermediate.
  • the magnesium salt is selected from one or a mixture of two of magnesium chloride and magnesium sulfate.
  • the molar concentration of the magnesium salt is 0.005mmol/L to 0.05mmol/L
  • the content of the deoxyribonuclease is 5KU/L to 50KU/L
  • the content of the ribonuclease is 5mg/L to 20mg/L
  • the content of the lipase is 1KU/L to 10KU/L.
  • phosphate buffered saline solution is used for cleaning first, and then ethanol is used for cleaning.
  • the nutrient solution is a DMEM/F12 culture solution containing fetal bovine serum.
  • the culture medium includes DMEM culture medium.
  • the DMEM culture solution is supplemented with fetal bovine serum, penicillin, streptomycin, amphotericin, glutamine, vitamin additives and vitamin C.
  • the liquid level of the culture solution is higher than the surface of the omentum acellular matrix material, and the height difference is less than 1 mm.
  • Figure 1 is a flow chart of the preparation method of the omentum acellular matrix material provided by the present invention
  • Figure 2 is a flow chart of the method for constructing cartilage tissue provided by the present invention.
  • Fig. 3 is a flowchart of a method for constructing cartilage tissue according to another embodiment of the present invention.
  • the preparation method includes the steps:
  • PBS phosphate buffered saline solution
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • tissue after the freeze-thaw cycle treatment is added to a solution containing 0.1% by mass trypsin, 0.01% by mass ethylenediaminetetraacetic acid (EDTA), and 0.1% by mass phenylmethylsulfonyl fluoride (PMSF). Shake for 6 hours at 30°C;
  • PBS phosphate buffered saline solution
  • the tissue obtained in the foregoing steps was added to the tissue containing 55mmol/L Na 2 HPO 4 , 17mmol/L KH 2 PO 4 , 1mmol/L MgSO 4 *7H 2 O, 5000 U/L deoxyribonuclease II (bovine pancreas), 5 mg/L
  • a solution of L ribonuclease IIIA (bovine pancreas) and 1000 U/L lipase VI-S (pig pancreas) shake at 30°C for 10 hours, and then use phosphate buffered saline solution PBS and 70% ethanol successively. Wash 3 times.
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • Tris 20mmol/L tris
  • EDTA 8mmol/L ethylenediaminetetraacetic acid
  • PMSF phenylmethylsulfonyl fluoride
  • PBS phosphate buffered saline solution
  • the tissue obtained in the foregoing steps was added to the tissue containing 55mmol/L Na 2 HPO 4 , 17mmol/L KH 2 PO 4 , 3mmol/L MgSO 4 *7H 2 O, 50000 U/L deoxyribonuclease II (bovine pancreas), 20 mg/L
  • 50000 U/L deoxyribonuclease II (bovine pancreas)
  • 20 mg/L In a solution of L ribonuclease IIIA (bovine pancreas) and 10000 U/L lipase VI-S (pig pancreas), shake at 40°C for 32 hours, and then use phosphate buffered saline solution PBS and 70% ethanol successively. Wash 3 times.
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • the present application also provides a method for constructing cartilage tissue, which includes the following steps:
  • this step can amplify the number of cartilage cells
  • step S20 Take a cell suspension from the culture solution obtained in step S10, and inoculate it on the omentum acellular matrix material prepared by the above-mentioned preparation method;
  • the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid surface is away from the omentum acellular matrix material.
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 5% by mass fetal bovine serum, 800IU/ml penicillin, 0.8mg/ml streptomycin, 2 ⁇ g/ml amphotericin, 1mmol/L glutamine, A DMEM culture solution containing 0.5% vitamin additives and 30 ⁇ g/ml vitamin C by weight.
  • the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid level is away from the omentum acellular matrix material.
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 15% fetal bovine serum by mass, 1200IU/ml penicillin, 1.2mg/ml streptomycin, 3 ⁇ g/ml amphotericin, 3mmol/L glutamine, DMEM culture solution containing 1.5% vitamin additives and 70 ⁇ g/ml vitamin C by weight.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • the inoculated omentum acellular matrix material in the culture medium for 4 days, change the culture medium once a day, the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid level is away from the omentum acellular matrix material
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 10% fetal bovine serum by mass, 1000IU/ml penicillin, 1mg/ml streptomycin, 2.5 ⁇ g/ml amphotericin, 2mmol/L glutamine, DMEM culture solution containing 1% vitamin additive and 50 ⁇ g/ml vitamin C.
  • this application also provides a method for constructing cartilage tissue, which includes the following steps:
  • step S09 taking a cell suspension from the culture solution obtained in step S08, and seeding it on the omentum acellular matrix material obtained in step S07;
  • PBS phosphate buffered saline solution
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid surface is away from the omentum acellular matrix material.
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 5% by mass fetal bovine serum, 800IU/ml penicillin, 0.8mg/ml streptomycin, 2 ⁇ g/ml amphotericin, 1mmol/L glutamine, A DMEM culture solution containing 0.5% vitamin additives and 30 ⁇ g/ml vitamin C by weight.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • tissue after the freeze-thaw cycle treatment is added to a solution containing 0.1% by mass trypsin, 0.01% by mass ethylenediaminetetraacetic acid (EDTA), and 0.1% by mass phenylmethylsulfonyl fluoride (PMSF). Shake for 6 hours at 30°C;
  • PBS phosphate buffered saline solution
  • the tissue obtained in the foregoing steps was added to the tissue containing 55mmol/L Na 2 HPO 4 , 17mmol/L KH 2 PO 4 , 1mmol/L MgSO 4 *7H 2 O, 5000 U/L deoxyribonuclease II (bovine pancreas), 5 mg/L
  • a solution of L ribonuclease IIIA (bovine pancreas) and 1000 U/L lipase VI-S (pig pancreas) shake at 30°C for 10 hours, and then use phosphate buffered saline solution PBS and 70% ethanol successively. Wash 3 times.
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid level is away from the omentum acellular matrix material.
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 15% fetal bovine serum by mass, 1200IU/ml penicillin, 1.2mg/ml streptomycin, 3 ⁇ g/ml amphotericin, 3mmol/L glutamine, DMEM culture solution containing 1.5% vitamin additives and 70 ⁇ g/ml vitamin C by weight.
  • Tris 20mmol/L tris
  • EDTA 8mmol/L ethylenediaminetetraacetic acid
  • PMSF phenylmethylsulfonyl fluoride
  • PBS phosphate buffered saline solution
  • the tissue obtained in the foregoing steps was added to the tissue containing 55mmol/L Na 2 HPO 4 , 17mmol/L KH 2 PO 4 , 3mmol/L MgSO 4 *7H 2 O, 50000 U/L deoxyribonuclease II (bovine pancreas), 20 mg/L
  • 50000 U/L deoxyribonuclease II (bovine pancreas)
  • 20 mg/L In a solution of L ribonuclease IIIA (bovine pancreas) and 10000 U/L lipase VI-S (pig pancreas), shake at 40°C for 32 hours, and then use phosphate buffered saline solution PBS and 70% ethanol successively. Wash 3 times.
  • the tissue obtained in the above steps is freeze-dried and irradiated to sterilize to obtain the omentum acellular matrix material.
  • the inoculated omentum acellular matrix material in the culture medium for 4 days, change the culture medium once a day, the liquid level of the culture medium is higher than the omentum acellular matrix material and the liquid level is away from the omentum acellular matrix material
  • the distance between the materials is less than 1mm, and the culture medium is supplemented with 10% fetal bovine serum by mass, 1000IU/ml penicillin, 1mg/ml streptomycin, 2.5 ⁇ g/ml amphotericin, 2mmol/L glutamine, DMEM culture solution containing 1% vitamin additive and 50 ⁇ g/ml vitamin C.

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Abstract

一种大网膜脱细胞基质材料的制备方法及软骨组织的构建方法。制备方法包括步骤：对大网膜进行清洗，进行冻融循环处理得到第一中间物，将第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、pH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物，对第二中间物进行脱脂处理，然后清洗得到第三中间物，将第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、pH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物，对第四中间物进行脱组织细胞处理，然后清洗得到第五中间物，将第五中间物进行干燥和灭菌处理，得到大网膜脱细胞基质材料。所述制备方法制备的大网膜脱细胞基质材料尤其适用于软骨组织的再生培养。

Description

大网膜脱细胞基质材料的制备方法及软骨组织的构建方法 技术领域
本发明涉及组织工程技术领域，具体涉及一种大网膜脱细胞基质材料的制备方法及软骨组织的构建方法。
背景技术
创伤、骨性关节炎等均为临床上常见的骨科疾病，这种疾病发病率较高，诱因也相对较多，且多数患者损伤后伴有软骨下骨的缺损，缺损如果得不到及时有效的修复，将会诱发其他疾病，严重者甚至导致关节功能的丧失。目前，临床上对于骨关节软骨缺损尚缺乏理想的修复方法，常用的修复方法尚存在一定的局限性，传统的软骨损伤治疗方法有关节磨削成形术、钻孔、微骨折及关节镜灌洗术，这些方法均不能将损坏的软骨及软骨下骨质修复成它们原来所具有的正常的组织结构。
大网膜是腹膜的一种。腹膜是存在高等脊椎动物腹腔中的一层黏膜，主要由间皮细胞构成，借由结缔组织的支持所形成的膜状组织。腹膜包覆大部分腹腔内的器官，能分泌黏液润湿脏器的表面，减轻脏器间的摩擦。腹膜从壁层向脏层移行，或从一器官移行于另一器官，构成双层的结构。两层腹膜间常有血管、神经和淋巴管走行。这些形成物依其本身结构特点和特定脏器联系而分别命名为韧带、网膜和系膜。大网膜是连接胃大弯至横结肠的腹膜，共四层：包括胃前、后壁的腹膜在胃大弯处愈合，形成大网膜的前两层，向下延伸至脐平面稍下方，然后向后上折返，包被横结肠，形成大网膜的后两层。大网膜是很薄的组织，富有极强的弹性和灵活性，同时具有高度血管化的结构，这些特点使其成为组织工程和再生医学领域中优良的天然生物材料。
目前，在组织工程研究领域，大网膜可作为体外移植构建物的“天然生物反应器”，促进构建物血管化的形成，增强移植构建物的功能，已成功应用于心肌组织工程、神经组织工程等领域。然而，目前尚未见将大网膜脱细胞基质应用于软骨组织工程的研究报道。
发明内容
基于上述现状，本发明的主要目的在于提供一种大网膜脱细胞基质材料的制备方法及软骨组织的构建方法，以解决现有大网膜脱细胞基质材料存在的上述问题。
为实现上述目的，本发明采用的技术方案如下：
本发明的第一方面提供了一种大网膜脱细胞基质材料的制备方法，所述制备方法包括步骤：
S100、对大网膜进行清洗；
S200、对清洗后的大网膜进行冻融循环处理得到第一中间物；
S300、将所述第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物，PH值例如可以为7.8、7.9、8、8.1、8.2，进一步优选为8；
S400、对所述第二中间物进行脱脂处理，然后清洗得到第三中间物；
S500、将所述第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物，PH值例如可以为7.8、7.9、8、8.1、8.2，进一步优选为8；
S600、对所述第四中间物进行脱组织细胞处理，然后清洗得到第五中间物；
S700、将所述第五中间物进行干燥和灭菌处理，得到所述大网膜脱细胞基质材料。
首先对大网膜进行冻融循环处理，在冻融循环过程中形成结冰以及进行冻融循环的盐溶液的浓度变化(冻融循环的冷冻过程中随着温度下降会导致未结冰水中的盐的溶解度出现变化，盐浓度逐渐上升，产生细胞膜内外浓度差)使得细胞破裂，从而提高细胞裂解效果，获得更大的总表面积以适于软骨组织的组织再生。
在脱脂处理和脱组织细胞处理之前，都要进行前处理过程，即在PH值7.8至8.2的液体环境下利用胰蛋白酶将赖氨酸与精氨酸残基中的羧基切断，从而将细胞打散，以利于后面的脱细胞处理，另外，由于在后续的脱细胞过程中，许多蛋白酶会从细胞内释放到处理液中，会对大网膜组织的其他蛋白质结构(构成基质材料的结构)造成破坏，因此，在前处理过程的溶液中加入了苯甲 基磺酰氟，苯甲基磺酰氟作为蛋白酶抑制剂，可以阻止前述的破坏过程，从而保证生成的基质材料的三维空间更加稳固，另外，采用这种方法生成的基质材料的三维空间可以促进软骨细胞的增殖与迁移、提高软骨细胞按照其原始表型表达的稳定性。
先进行前处理，再进行脱细胞处理，能够提高基质微粒的表面粗糙度，从而更有利于软骨细胞的黏附，以利于软骨组织的再生。
大网膜可以选用猪、牛、羊等哺乳动物的大网膜，其可以从屠宰场等场所购买得到。
优选地，在所述步骤S100中，采用生理盐水对所述大网膜进行清洗。
生理盐水具有一定的杀菌灭菌效果，可使细菌产生胞内外浓度差，进而易使细菌细胞内产生质壁分离的效果，抑制细菌的繁殖，另外，生理盐水对生理细胞无伤害，以保证大网膜的完整性。清洗次数可以为2-3次。
优选地，在所述步骤S200中，将清洗后的大网膜加入到含有三羟甲基氨基甲烷和苯甲基磺酰氟且PH值为7.8至8.2的缓冲液中进行所述冻融循环。
采用上述缓冲液一方面能够提高细胞裂解效果，另一方面苯甲基磺酰氟的添加能够为组织提供一个稳定的环境，避免细胞内释放的蛋白酶对大网膜组织的其他蛋白质结构(构成基质材料的结构)造成破坏，从而保证生成的基质材料的三维空间更加稳固。
优选地，所述缓冲液中，所述三羟甲基氨基甲烷的摩尔浓度为1mmol/L至20mmol/L，例如可以为1mmol/L、2mmol/L、3mmol/L、4mmol/L、5mmol/L、6mmol/L、7mmol/L、8mmol/L、9mmol/L、10mmol/L、11mmol/L、12mmol/L、13mmol/L、14mmol/L、15mmol/L、16mmol/L、17mmol/L、18mmol/L、19mmol/L、20mmol/L，进一步优选为8mmol/L至12mmol/L，更进一步优选为10mmol/L，所述苯甲基磺酰氟的质量百分比为0.1％至1％，例如质量百分比为0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％，进一步优选为0.8％至1％，更进一步优选为1％。
优选地，所述缓冲液中还含有乙二胺四乙酸，所述乙二胺四乙酸的摩尔浓度为1mmol/L至10mmol/L，例如为1mmol/L、2mmol/L、3mmol/L、4mmol/L、5mmol/L、6mmol/L、7mmol/L、8mmol/L、9mmol/L、10mmol/L，进一步优选为8mmol/L至10mmol/L，更进一步优选为10mmol/L。
利用乙二胺四乙酸来调节缓冲液的PH值，使其PH值控制在7.8至8.2范围内，另外，由于乙二胺四乙酸具有抑制作用，采用其进行PH值调节能够避免因引入新的物质而对大网膜组织的其他蛋白质结构(构成基质材料的结构)造成破坏，从而进一步保证生成的基质材料的三维空间更加稳固。
优选地，所述冻融循环中一次冻融循环包括：首先在-80℃至-20℃温度条件下进行1至8小时的冷冻处理，冷冻处理的温度例如可以为-80℃、-75℃、-70℃、-65℃、-60℃、-55℃、-50℃、-45℃、-40℃、-35℃、-30℃、-25℃、-20℃，冷冻处理的时长例如可以为1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时；然后在30℃至40℃温度条件下进行0.5至1小时的融化处理，融化处理的温度例如可以为30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃，融化处理的时长例如可以为0.5小时、0.6小时、0.7小时、0.8小时、0.9小时、1小时。
优选地，冻融循环的次数可以为2-3次，进一步优选为3次。
冻融循环虽然是一种现有的处理技术，但由于大网膜脱细胞基质材料的要求非常苛刻，融化温度过高会导致蛋白质变性，融化温度过低会导致融解不完全，因苯甲基磺酰氟这种蛋白酶抑制剂半衰期太短，如若融化处理时间过长，会导致细胞裂解后的细胞内蛋白酶对细胞外基质酶解，因此，现有的大网膜脱细胞基质材料的制备过程中并没有采用这一技术，本申请中，通过对温度和时长的限制，使得冻融循环能够很好的使得细胞破裂且不会对细胞外基质造成破坏。
优选地，在所述步骤S300中，所述胰蛋白酶的质量百分比为0.1％至0.5％，例如为0.1％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％、0.45％、0.5％，进一步优选为0.25％，所述苯甲基磺酰氟的质量百分比为0.1％至1％，例如为0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％，进一步优选为0.8％至1％，更进一步优选为1％；
在所述步骤S500中，所述胰蛋白酶的质量百分比为0.1％至0.5％，例如为0.1％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％、0.45％、0.5％，进一步优选为0.25％，所述苯甲基磺酰氟的质量百分比为0.1％至1％，例如为0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％，进一步优选为0.8％至1％，更进一步优选为1％。
优选地，所述步骤S300和所述步骤S500中的溶液中均添加有乙二胺四乙酸，且乙二胺四乙酸的质量百分比为0.01％至0.1％，例如为0.01％、0.02％、0.03％、0.04％、0.05％、0.06％、0.07％、0.08％、0.09％、0.1％，进一步优选为0.8％至0.1％，更进一步优选为0.1％。
在步骤S300和步骤S500中，利用乙二胺四乙酸来调节缓冲液的PH值，使其PH值控制在7.8至8.2范围内，另外，由于乙二胺四乙酸具有抑制作用，采用其进行PH值调节能够避免因引入新的物质而对大网膜组织的其他蛋白质结构(构成基质材料的结构)造成破坏，从而进一步保证生成的基质材料的三维空间更加稳固。
优选地，所述第一预定时长为6至12小时，例如为6小时、7小时、8小时、9小时、10小时、11小时、12小时；进一步优选地，步骤S300中，将第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，振荡并持续第一预定时长。
所述第二预定时长为3至6小时，例如为3小时、4小时、5小时、6小时。进一步优选地，步骤S500中，将第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，振荡并持续第二预定时长。
优选地，在所述步骤S400中，采用异丙醇、正丁醇、乙腈、乙醇、甲醇中的一种或至少两种的混合物对所述第二中间物进行脱脂处理。进一步优选采用异丙醇对第二中间物进行脱脂处理，异丙醇的相对于甲醇、氯仿、乙醚来说沸点高、无刺激性气味，不易挥发，能够减少对环境的污染。进一步优选地，步骤S400中，将第二中间物放入异丙醇、正丁醇、乙腈、乙醇、甲醇中的一种或至少两种的混合物中，在恒温条件(30℃至40℃，例如30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃)下振荡40至50小时，例如40小时、41小时、42小时、43小时、44小时、45小时、46小时、47小时、48小时、49小时、50小时，进一步优选为48小时。
优选地，在所述步骤S400中，采用磷酸缓冲盐溶液进行清洗，清洗次数为2-3次，磷酸缓冲盐溶液的PH值优选为7.8至8.2，例如为7.8、7.9、8、8.1、8.2，进一步优选为8。
优选地，在所述步骤S600中，采用含有镁盐、脱氧核糖核酸酶、核糖核 酸酶以及脂肪酶的溶液对所述第四中间物进行脱组织细胞处理。进一步优选地，在步骤S600中，将第四中间物放入含有镁盐、脱氧核糖核酸酶、核糖核酸酶以及脂肪酶的溶液中恒温条件(30℃至40℃，例如30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃)下振荡10至32小时，例如10小时、12小时、14小时、16小时、18小时、20小时、22小时、24小时、26小时、28小时、30小时、32小时，进一步优选为14至18小时，更进一步优选为16小时。
在步骤S600中，采用含有脱氧核糖核酸酶、核糖核酸酶以及脂肪酶的溶液对第四中间物进行脱组织细胞处理，配合步骤S500的前处理过程使得细胞离散，能够有效除去所有细胞成分，镁盐中的二价镁离子有抑制胰蛋白酶的作用，使得脱组织细胞处理不会对基质材料破坏，保证基质材料的完整性。
优选地，所述镁盐为氯化镁、硫酸镁中的一种或两者的混合物。
优选地，所述镁盐的摩尔浓度为0.005mmol/L至0.05mmol/L，例如为0.005mmol/L、0.01mmol/L、0.015mmol/L、0.02mmol/L、0.025mmol/L、0.03mmol/L、0.035mmol/L、0.04mmol/L、0.045mmol/L、0.05mmol/L，所述脱氧核糖核酸酶的含量为5KU/L至50KU/L，例如为5KU/L、10KU/L、15KU/L、20KU/L、25KU/L、30KU/L、35KU/L、40KU/L、45KU/L、50KU/L，进一步优选为10KU/L至20KU/L，更进一步优选为15KU/L，脱氧核糖核酸酶的酶类型为Type II、Type II-S或Type IV，所述核糖核酸酶的含量为5mg/L至20mg/L，例如为5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、10.5mg/L、11mg/L、11.5mg/L、12mg/L、12.5mg/L、13mg/L、13.5mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L、20mg/L，进一步优选为10mg/L至14mg/L，更进一步优选为12.5mg/L，核糖核酸酶的酶类型为Type I-A、Type I-AS、Type II-A、Type III-A、Type X-A、Type XII-A，所述脂肪酶的含量为1KU/L至10KU/L，例如为1KU/L、2KU/L、3KU/L、4KU/L、5KU/L、6KU/L、7KU/L、8KU/L、9KU/L、10KU/L，进一步优选为1KU/L至4KU/L，更进一步优选为2KU/L，脂肪酶的酶类型为Type II、VI-S。
优选地，在所述步骤S600中，首先采用磷酸缓冲盐溶液进行清洗，磷酸缓冲盐溶液的PH值优选为7.8至8.2，例如为7.8、7.9、8、8.1、8.2，进一步优选为8，清洗次数可以为2-3次，然后再采用乙醇进行清洗，乙醇浓度优选 为50％至70％，清洗次数可以为2-3次。
优选地，在所述步骤S700中，采用冻干方式进行干燥，采用辐照灭菌方式进行灭菌处理。
优选地，在所述步骤S500中，得到的所述第四中间物采用PH值7.8至8.2的磷酸缓冲盐溶液进行清洗后再进入步骤S600。
综上，采用本申请的方法能够完整地保留细胞外基质的三维空间结构及一些对细胞分化有重要作用的生长因子(例如转化生长因子(TGF-β)、碱性成纤维细胞生长因子(bFGF)等生长因子)，采用该方法制备的大网膜脱细胞基质材料构成的三维空间可以促进软骨细胞的增殖与迁移、提高软骨细胞按照其原始表型表达的稳定性，促进细胞基质的分泌从而增加支架的坚固性。此外，本申请制备的大网膜脱细胞基质材料还具有良好的生物相容性、细胞吸附性和亲水性，且降解性能和宿主整合能力强。
本发明的第二方面提供了一种软骨组织的构建方法，所述构建方法包括步骤：
S10、将用于软骨组织构建的种子细胞置于营养液中，并持续第三预定时长；采用该步骤可以扩增软骨细胞的数量；
S20、从所述步骤S10得到的培养液中取细胞悬液，并接种于采用如上所述的制备方法制备的大网膜脱细胞基质材料上；
S30、将接种后的大网膜脱细胞基质材料置于培养液中进行培养，并持续第四预定时长，得到构建的软骨组织。
本申请提供的构建方法，将种子细胞吸附于生物相容性良好并可以为人体逐渐吸收的大网膜脱细胞基质材料构成的支架载体中，该载体为细胞提供一个生存的三维空间，有利于细胞获得足够的营养物质，生长，扩增，从而获得构建好的软骨组织，构建好的软骨组织与支架载体形成一个整体的移植物，移植物植入机体内的组织病损部位，种植的细胞继续增殖并分泌基质，形成新的具有原位组织细胞特殊形态和功能的相应软骨组织，而植入部位周围的结缔组织细胞将生长进入大网膜脱细胞基质材料的网状结构中，如此，逐渐形成与正常组织类似的仿生结构，从而起到修复作用了，患者可以通过定制、移植自体软骨移植物获得软骨缺损的修复和功能再造，实现完全治愈和持久的康复。
步骤S10中，用于软骨组织构建的种子细胞可以在细胞库进行购买，也可 以取患者或者志愿者的软骨组织，在取患者的软骨组织时，优选取非承重区软骨细胞丰富的软骨组织，当取患者的软骨组织时，可以在无菌条件下取非承重区域的软骨组织，放入盛有营养液的培养瓶中洗涤2至3次，然后在无菌条件下将软骨组织剪成小块并用消毒滤纸吸干，放入盛有营养液的培养瓶中。
在所述步骤S10中，所述营养液为含有胎牛血清的DMEM/F12培养液，DMEM(dulbecco's modified eagle medium)是一种含各种氨基酸和葡萄糖的培养基，F12(Ham‘s F 12 nutrient medium)是一种动物细胞培养基，DMEM/F12培养液由DMEM和F12以1:1结合得到。第三预定时长优选为6-8周。
优选地，在所述步骤S30中，所述培养液包括DMEM培养液。由于DMEM含有多种氨基酸和葡萄糖，大大提高了细胞的黏附率。
优选地，所述DMEM培养液中添加有胎牛血清、青霉素、链霉素、两性霉素、谷氨酰胺、维生素添加剂和维生素C。其中，胎牛血清的质量百分比为5％至15％，例如5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％，进一步优选为10％，青霉素的含量为800IU/ml至1200IU/ml，例如800IU/ml、900IU/ml、1000IU/ml、1100IU/ml、1200IU/ml，进一步优选为1000IU/ml，链霉素的含量为0.8mg/ml至1.2mg/ml，例如为0.8mg/ml、0.9mg/ml、1mg/ml、1.1mg/ml、1.2mg/ml，进一步优选为1mg/ml，两性霉素的含量为2μg/ml至3μg/ml，例如为2μg/ml、2.1μg/ml、2.2μg/ml、2.3μg/ml、2.4μg/ml、2.5μg/ml、2.6μg/ml、2.7μg/ml、2.8μg/ml、2.9μg/ml、3μg/ml，进一步优选为2.5μg/ml，谷氨酰胺的含量为1mmol/L至3mmol/L，例如为1mmol/L、1.5mmol/L、2mmol/L、2.5mmol/L、3mmol/L，进一步优选为2mmol/L，维生素添加剂的质量百分比为0.5％至1.5％，例如0.5％、0.6％、0.7％、0.8％、0.9％、1％、1.1％、1.2％、1.3％、1.4％、1.5％，进一步优选为1％，维生素C的含量为30μg/ml至70μg/ml，例如为30μg/ml、35μg/ml、40μg/ml、45μg/ml、50μg/ml、55μg/ml、60μg/ml、65μg/ml、70μg/ml，进一步优选为50μg/ml。
其中，胎牛血清为细胞提供必需的营养物质、对培养基进行酸碱平衡、解毒以及抑制某些酶制剂的活性；青霉素、链霉素、两性霉素主要是消除污染，软骨细胞培养过程中需要考虑两种污染：微生物污染和其他细胞系的混入，两种污染都非常普遍，必须重视可能的污染以及污染对结果造成的影响，因此使 用抗生素和抗真菌剂来防止污染；谷氨酰胺在细胞培养时是重要的，脱掉氨基后，L-谷氨酰胺可作为培养细胞的能量来源、参与蛋白质的合成和核酸代谢；维生素添加剂提供营养作用；维生素C对细胞起到抗氧化保护作用。
优选地，第四预定时长优选为3-5天。
优选地，所述培养液的液面高于所述大网膜脱细胞基质材料的表面，且高度差小于1mm。
将培养液的液面设置为略高于大网膜脱细胞基质材料的表面，能够使得种植了细胞的大网膜脱细胞基质材料与含有5％左右CO 2的空气接触，保证气-液面培养，从而提高培养效率以及保证培养效果。
优选地，步骤S30的培养温度在35℃至38℃之间，优选为37℃，且优选每天进行一次培养液的更换。
本发明还提供了一种软骨组织的构建方法，所述构建方法包括步骤：
S01、对大网膜进行清洗；
S02、对清洗后的大网膜进行冻融循环处理得到第一中间物；
S03、将所述第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物；
S04、对所述第二中间物进行脱脂处理，然后清洗得到第三中间物；
S05、将所述第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物；
S06、对所述第四中间物进行脱组织细胞处理，然后清洗得到第五中间物；
S07、将所述第五中间物进行干燥和灭菌处理，得到所述大网膜脱细胞基质材料；
S08、将用于软骨组织构建的种子细胞置于营养液中，并持续第三预定时长；
S09、从步骤S08得到的培养液中取细胞悬液，并接种于步骤S07得到的大网膜脱细胞基质材料上；
S010、将接种后的大网膜脱细胞基质材料置于培养液中进行培养，并持续第四预定时长，得到构建的软骨组织。
制备的大网膜脱细胞基质材料由于具有较大的总表面积且三维空间非常稳固，非常适于后续的软骨组织的再生培养，促进软骨细胞的增殖与迁移、提 高软骨细胞按照其原始表型表达的稳定性。
优选地，在所述步骤S01中，采用生理盐水对所述大网膜进行清洗。
优选地，在所述步骤S02中，将清洗后的大网膜加入到含有三羟甲基氨基甲烷和苯甲基磺酰氟且PH值为7.8至8.2的缓冲液中进行所述冻融循环。
优选地，所述缓冲液中，所述三羟甲基氨基甲烷的摩尔浓度为1mmol/L至20mmol/L，所述苯甲基磺酰氟的质量百分比为0.1％至1％。
优选地，所述缓冲液中还含有乙二胺四乙酸，所述乙二胺四乙酸的摩尔浓度为1mmol/L至10mmol/L。
优选地，所述冻融循环中一次冻融循环包括：首先在-80℃至-20℃温度条件下进行1至8小时的冷冻处理，然后在30℃至40℃温度条件下进行0.5至1小时的融化处理。
优选地，在所述步骤S03中，所述胰蛋白酶的质量百分比为0.1％至0.5％，所述苯甲基磺酰氟的质量百分比为0.1％至1％；
在所述步骤S05中，所述胰蛋白酶的质量百分比为0.1％至0.5％，所述苯甲基磺酰氟的质量百分比为0.1％至1％。
优选地，所述步骤S03和步骤S05中的溶液中均添加有乙二胺四乙酸，且乙二胺四乙酸的质量百分比为0.01％至0.1％。
优选地，在所述步骤S04中，采用异丙醇、正丁醇、乙腈、乙醇、甲醇中的一种或至少两种的混合物对所述第二中间物进行脱脂处理。
优选地，在所述步骤S04中，采用磷酸缓冲盐溶液进行清洗。
优选地，在所述步骤S06中，采用含有镁盐、脱氧核糖核酸酶、核糖核酸酶以及脂肪酶的溶液对所述第四中间物进行脱组织细胞处理。
优选地，所述镁盐选自氯化镁、硫酸镁中的一种或两种的混合物。
优选地，所述镁盐的摩尔浓度为0.005mmol/L至0.05mmol/L，所述脱氧核糖核酸酶的含量为5KU/L至50KU/L，所述核糖核酸酶的含量为5mg/L至20mg/L，所述脂肪酶的含量为1KU/L至10KU/L。
优选地，在所述步骤S06中，首先采用磷酸缓冲盐溶液进行清洗，然后再采用乙醇进行清洗。
优选地，在所述步骤S08中，所述营养液为含有胎牛血清的DMEM/F12培养液。
优选地，在所述步骤S010中，所述培养液包括DMEM培养液。
优选地，所述DMEM培养液中添加有胎牛血清、青霉素、链霉素、两性霉素、谷氨酰胺、维生素添加剂和维生素C。
优选地，所述培养液的液面高于所述大网膜脱细胞基质材料的表面，且高度差小于1mm。
附图说明
以下将参照附图对本发明的优选实施方式进行描述。图中：
图1为本发明提供的大网膜脱细胞基质材料的制备方法的流程图；
图2为本发明提供的软骨组织的构建方法流程图；
图3为本发明提供的另一实施例的软骨组织的构建方法流程图。
具体实施方式
以下基于实施例对本发明进行描述，但是本发明并不仅仅限于这些实施例。在下文对本发明的细节描述中，详尽描述了一些特定的细节部分，为了避免混淆本发明的实质，公知的方法、过程、流程、元件并没有详细叙述。
此外，本领域普通技术人员应当理解，在此提供的附图都是为了说明的目的，并且附图不一定是按比例绘制的。
除非上下文明确要求，否则整个说明书和权利要求书中的“包括”、“包含”等类似词语应当解释为包含的含义而不是排他或穷举的含义；也就是说，是“包括但不限于”的含义。
在本发明的描述中，需要理解的是，术语“第一”、“第二”等仅用于描述目的，而不能理解为指示或暗示相对重要性。此外，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
由于软骨组织的自身特质，采用现有方法制备的大网膜脱细胞基质材料并不适于软骨组织的再生培养，基于此，本申请提供了一种大网膜脱细胞基质材料的制备方法，当然，可以理解的是，本申请提供的制备方法制备的大网膜脱细胞基质材料尤其适用于软骨组织的再生培养，当然，其也可以应用于其他组织的再生培养。如图1所示，该制备方法包括步骤：
S100、对大网膜进行清洗；
S200、对清洗后的大网膜进行冻融循环处理得到第一中间物；
S300、将所述第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物；
S400、对所述第二中间物进行脱脂处理，然后清洗得到第三中间物；
S500、将所述第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物；
S600、对所述第四中间物进行脱组织细胞处理，然后清洗得到第五中间物；
S700、将所述第五中间物进行干燥和灭菌处理，得到所述大网膜脱细胞基质材料。
下面给出该制备方法的具体实施例：
实施例一：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含10mmol/L三羟甲基氨基甲烷(Tris)、10mmol/L乙二胺四乙酸(EDTA)以及质量百分比1％苯甲基磺酰氟(PMSF)的pH＝8.0的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-80℃，持续时长为1小时，融化温度为37℃，持续时长为0.5小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.25％胰蛋白酶、质量百分比0.1％乙二胺四乙酸(EDTA)、质量百分比1％苯甲基磺酰氟(PMSF)的溶液中，在37℃条件下振荡12小时；
将前述步骤得到的组织加入到异丙醇中在37℃条件下振荡脱脂48小时，再用pH＝8.0的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.25％胰蛋白酶、质量百分比0.1％乙二胺四乙酸(EDTA)、质量百分比1％苯甲基磺酰氟(PMSF)的溶液中，在37℃条件下振荡6小时，再用pH＝8.0的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/LNa 2HPO 4、17mmol/LKH 2PO 4、4.9mmol/LMgSO 4*7H 2O、15000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、12.5mg/L核糖核酸酶IIIA(牛胰脏)、2000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在37℃条件下振荡16小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材 料。
实施例二：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含1mmol/L三羟甲基氨基甲烷(Tris)、1mmol/L乙二胺四乙酸(EDTA)以及质量百分比0.1％苯甲基磺酰氟(PMSF)的pH＝7.8的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-20℃，持续时长为8小时，融化温度为30℃，持续时长为1小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.1％胰蛋白酶、质量百分比0.01％乙二胺四乙酸(EDTA)、质量百分比0.1％苯甲基磺酰氟(PMSF)的溶液中，在30℃条件下振荡6小时；
将前述步骤得到的组织加入到异丙醇中在30℃条件下振荡脱脂40小时，再用pH＝7.8的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.1％胰蛋白酶、质量百分比0.01％乙二胺四乙酸(EDTA)、质量百分比0.1％苯甲基磺酰氟(PMSF)的溶液中，在30℃条件下振荡3小时，再用pH＝7.8的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/L Na 2HPO 4、17mmol/L KH 2PO 4、1mmol/L MgSO 4*7H 2O、5000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、5mg/L核糖核酸酶IIIA(牛胰脏)、1000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在30℃条件下振荡10小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材料。
实施例三：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含20mmol/L三羟甲基氨基甲烷(Tris)、8mmol/L乙二胺四乙酸(EDTA)以及质量百分比0.8％苯甲基磺酰氟(PMSF)的pH＝8.2的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-50℃，持续时长为5小时，融化温度为40℃，持续时长为0.8小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.5％胰蛋白酶、质量百分比0.08％乙二胺四乙酸(EDTA)、质量百分比0.8％苯甲基磺酰氟 (PMSF)的溶液中，在40℃条件下振荡12小时；
将前述步骤得到的组织加入到异丙醇中在40℃条件下振荡脱脂50小时，再用pH＝8.2的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.5％胰蛋白酶、质量百分比0.08％乙二胺四乙酸(EDTA)、质量百分比0.8％苯甲基磺酰氟(PMSF)的溶液中，在40℃条件下振荡6小时，再用pH＝8.2的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/L Na 2HPO 4、17mmol/L KH 2PO 4、3mmol/L MgSO 4*7H 2O、50000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、20mg/L核糖核酸酶IIIA(牛胰脏)、10000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在40℃条件下振荡32小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材料。
如图2所示，本申请还提供了一种软骨组织的构建方法，其包括如下步骤：
S10、将用于软骨组织构建的种子细胞置于营养液中，并持续第三预定时长；采用该步骤可以扩增软骨细胞的数量；
S20、从所述步骤S10得到的培养液中取细胞悬液，并接种于采用如上所述的制备方法制备的大网膜脱细胞基质材料上；
S30、将接种后的大网膜脱细胞基质材料置于培养液中进行培养，并持续第四预定时长，得到构建的软骨组织。
下面给出该构建方法的具体实施例：
实施例四：
将在细胞库购买的用于软骨组织构建的种子细胞置于含有胎牛血清的DMEM/F12培养液中培养6周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养5天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比5％胎牛血清、800IU/ml的青霉素、0.8mg/ml的链霉素、2μg/ml的两性霉素、1mmol/L的谷氨酰胺、质量百分比0.5％维生素添加剂、30μg/ml的维生素C的DMEM培养液。
实施例五：
在无菌条件下取患者非承重区的软骨组织，放入盛有营养液的培养瓶中洗涤2至3次，然后在无菌条件下将软骨组织剪成小块并用消毒滤纸吸干，放入含有胎牛血清的DMEM/F12培养液中培养8周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养3天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比15％胎牛血清、1200IU/ml的青霉素、1.2mg/ml的链霉素、3μg/ml的两性霉素、3mmol/L的谷氨酰胺、质量百分比1.5％维生素添加剂、70μg/ml的维生素C的DMEM培养液。
实施例六：
在无菌条件下取患者非承重区的软骨组织，放入盛有营养液的培养瓶中洗涤2至3次，然后在无菌条件下将软骨组织剪成小块并用消毒滤纸吸干，放入含有胎牛血清的DMEM/F12培养液中培养7周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养4天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比10％胎牛血清、1000IU/ml的青霉素、1mg/ml的链霉素、2.5μg/ml的两性霉素、2mmol/L的谷氨酰胺、质量百分比1％维生素添加剂、50μg/ml的维生素C的DMEM培养液。
如图3所示，本申请还提供了一种软骨组织的构建方法，其包括如下步骤：
S01、对大网膜进行清洗；
S02、对清洗后的大网膜进行冻融循环处理得到第一中间物；
S03、将所述第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物；
S04、对所述第二中间物进行脱脂处理，然后清洗得到第三中间物；
S05、将所述第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物；
S06、对所述第四中间物进行脱组织细胞处理，然后清洗得到第五中间物；
S07、将所述第五中间物进行干燥和灭菌处理，得到所述大网膜脱细胞基 质材料；
S08、将用于软骨组织构建的种子细胞置于营养液中，并持续第三预定时长；
S09、从步骤S08得到的培养液中取细胞悬液，并接种于步骤S07得到的大网膜脱细胞基质材料上；
S010、将接种后的大网膜脱细胞基质材料置于培养液中进行培养，并持续第四预定时长，得到构建的软骨组织。
下面给出该构建方法的具体实施例：
实施例七：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含10mmol/L三羟甲基氨基甲烷(Tris)、10mmol/L乙二胺四乙酸(EDTA)以及质量百分比1％苯甲基磺酰氟(PMSF)的pH＝8.0的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-80℃，持续时长为1小时，融化温度为37℃，持续时长为0.5小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.25％胰蛋白酶、质量百分比0.1％乙二胺四乙酸(EDTA)、质量百分比1％苯甲基磺酰氟(PMSF)的溶液中，在37℃条件下振荡12小时；
将前述步骤得到的组织加入到异丙醇中在37℃条件下振荡脱脂48小时，再用pH＝8.0的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.25％胰蛋白酶、质量百分比0.1％乙二胺四乙酸(EDTA)、质量百分比1％苯甲基磺酰氟(PMSF)的溶液中，在37℃条件下振荡6小时，再用pH＝8.0的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/LNa 2HPO 4、17mmol/LKH 2PO 4、4.9mmol/LMgSO 4*7H 2O、15000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、12.5mg/L核糖核酸酶IIIA(牛胰脏)、2000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在37℃条件下振荡16小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材料。
将在细胞库购买的用于软骨组织构建的种子细胞置于含有胎牛血清的 DMEM/F12培养液中培养6周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养5天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比5％胎牛血清、800IU/ml的青霉素、0.8mg/ml的链霉素、2μg/ml的两性霉素、1mmol/L的谷氨酰胺、质量百分比0.5％维生素添加剂、30μg/ml的维生素C的DMEM培养液。
实施例八：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含1mmol/L三羟甲基氨基甲烷(Tris)、1mmol/L乙二胺四乙酸(EDTA)以及质量百分比0.1％苯甲基磺酰氟(PMSF)的pH＝7.8的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-20℃，持续时长为8小时，融化温度为30℃，持续时长为1小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.1％胰蛋白酶、质量百分比0.01％乙二胺四乙酸(EDTA)、质量百分比0.1％苯甲基磺酰氟(PMSF)的溶液中，在30℃条件下振荡6小时；
将前述步骤得到的组织加入到异丙醇中在30℃条件下振荡脱脂40小时，再用pH＝7.8的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.1％胰蛋白酶、质量百分比0.01％乙二胺四乙酸(EDTA)、质量百分比0.1％苯甲基磺酰氟(PMSF)的溶液中，在30℃条件下振荡3小时，再用pH＝7.8的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/L Na 2HPO 4、17mmol/L KH 2PO 4、1mmol/L MgSO 4*7H 2O、5000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、5mg/L核糖核酸酶IIIA(牛胰脏)、1000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在30℃条件下振荡10小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材料。
在无菌条件下取患者非承重区的软骨组织，放入盛有营养液的培养瓶中洗涤2至3次，然后在无菌条件下将软骨组织剪成小块并用消毒滤纸吸干，放入 含有胎牛血清的DMEM/F12培养液中培养8周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养3天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比15％胎牛血清、1200IU/ml的青霉素、1.2mg/ml的链霉素、3μg/ml的两性霉素、3mmol/L的谷氨酰胺、质量百分比1.5％维生素添加剂、70μg/ml的维生素C的DMEM培养液。
实施例九：
将新鲜猪大网膜用生理盐水洗涤3次；
将完成洗涤的大网膜加入到含20mmol/L三羟甲基氨基甲烷(Tris)、8mmol/L乙二胺四乙酸(EDTA)以及质量百分比0.8％苯甲基磺酰氟(PMSF)的pH＝8.2的缓冲液中冻融循环3次，其中，一次冻融循环中冷冻温度为-50℃，持续时长为5小时，融化温度为40℃，持续时长为0.8小时；
将经过冻融循环处理后的组织加入到含有质量百分比0.5％胰蛋白酶、质量百分比0.08％乙二胺四乙酸(EDTA)、质量百分比0.8％苯甲基磺酰氟(PMSF)的溶液中，在40℃条件下振荡12小时；
将前述步骤得到的组织加入到异丙醇中在40℃条件下振荡脱脂50小时，再用pH＝8.2的磷酸缓冲盐溶液PBS(8g/L NaCl、200mg/L KCl、1g/L Na 2HPO 4、200mg/L KH 2PO 4)清洗3次。
将清洗后的组织加入到含有质量百分比0.5％胰蛋白酶、质量百分比0.08％乙二胺四乙酸(EDTA)、质量百分比0.8％苯甲基磺酰氟(PMSF)的溶液中，在40℃条件下振荡6小时，再用pH＝8.2的PBS清洗3次。
将前述步骤得到组织加入到含55mmol/L Na 2HPO 4、17mmol/L KH 2PO 4、3mmol/L MgSO 4*7H 2O、50000U/L脱氧核糖核酸酶Ⅱ(牛胰脏)、20mg/L核糖核酸酶IIIA(牛胰脏)、10000U/L脂肪酶Ⅵ-S(猪胰脏)的溶液中，在40℃条件下振荡32小时，再用磷酸缓冲盐溶液PBS和70％乙醇先后各清洗3次。
将上述步骤得到的组织进行冻干和辐照灭菌，得到大网膜脱细胞基质材料。
在无菌条件下取患者非承重区的软骨组织，放入盛有营养液的培养瓶中洗涤2至3次，然后在无菌条件下将软骨组织剪成小块并用消毒滤纸吸干，放入 含有胎牛血清的DMEM/F12培养液中培养7周；
取细胞悬液接种于大网膜脱细胞基质材料上；
将接种后的大网膜脱细胞基质材料置于培养液中培养4天，每天更换一次培养液，培养液的液面高于大网膜脱细胞基质材料且液面距离大网膜脱细胞基质材料的距离小于1mm，培养液为添加了质量百分比10％胎牛血清、1000IU/ml的青霉素、1mg/ml的链霉素、2.5μg/ml的两性霉素、2mmol/L的谷氨酰胺、质量百分比1％维生素添加剂、50μg/ml的维生素C的DMEM培养液。
本领域的技术人员能够理解的是，在不冲突的前提下，上述各优选方案可以自由地组合、叠加。
应当理解，上述的实施方式仅是示例性的，而非限制性的，在不偏离本发明的基本原理的情况下，本领域的技术人员可以针对上述细节做出的各种明显的或等同的修改或替换，都将包含于本发明的权利要求范围内。

Claims (10)

  1. 一种大网膜脱细胞基质材料的制备方法，其特征在于，所述制备方法包括步骤：
    S100、对大网膜进行清洗；
    S200、对清洗后的大网膜进行冻融循环处理得到第一中间物；
    S300、将所述第一中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第一预定时长得到第二中间物；
    S400、对所述第二中间物进行脱脂处理，然后清洗得到第三中间物；
    S500、将所述第三中间物加入含有胰蛋白酶和苯甲基磺酰氟、PH值为7.8至8.2的溶液中，并持续第二预定时长得到第四中间物；
    S600、对所述第四中间物进行脱组织细胞处理，然后清洗得到第五中间物；
    S700、将所述第五中间物进行干燥和灭菌处理，得到所述大网膜脱细胞基质材料。
  2. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S100中，采用生理盐水对所述大网膜进行清洗。
  3. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S200中，将清洗后的大网膜加入到含有三羟甲基氨基甲烷和苯甲基磺酰氟且PH值为7.8至8.2的缓冲液中进行所述冻融循环。
  4. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S300中，所述胰蛋白酶的质量百分比为0.1％至0.5％，所述苯甲基磺酰氟的质量百分比为0.1％至1％；
    在所述步骤S500中，所述胰蛋白酶的质量百分比为0.1％至0.5％，所述苯甲基磺酰氟的质量百分比为0.1％至1％。
  5. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S400中，采用异丙醇、正丁醇、乙腈、乙醇、甲醇中的一种或至少两种的混合物对所述第二中间物进行脱脂处理。
  6. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S600中，采用含有镁盐、脱氧核糖核酸酶、核糖核酸酶以及脂肪酶的溶液对所述第四中间物进行脱组织细胞处理。
  7. 根据权利要求1所述的大网膜脱细胞基质材料的制备方法，其特征在于，在所述步骤S600中，首先采用磷酸缓冲盐溶液进行清洗，然后再采用乙醇进行清洗。
  8. 一种软骨组织的构建方法，其特征在于，所述构建方法包括步骤：
    S10、将用于软骨组织构建的种子细胞置于营养液中，并持续第三预定时长；
    S20、从所述步骤S10得到的培养液中取细胞悬液，并接种于采用如权利要求1至7任一项所述的制备方法制备的大网膜脱细胞基质材料上；
    S30、将接种后的大网膜脱细胞基质材料置于培养液中进行培养，并持续第四预定时长，得到构建的软骨组织。
  9. 根据权利要求8所述的软骨组织的构建方法，其特征在于，在所述步骤S10中，所述营养液为含有胎牛血清的DMEM/F12培养液。
  10. 根据权利要求8或9所述的软骨组织的构建方法，其特征在于，所述培养液的液面高于所述大网膜脱细胞基质材料的表面，且高度差小于1mm。
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