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WO2024178882A1 - Procédé de préparation de membrane amniotique sèche et son utilisation - Google Patents

Procédé de préparation de membrane amniotique sèche et son utilisation Download PDF

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Publication number
WO2024178882A1
WO2024178882A1 PCT/CN2023/103200 CN2023103200W WO2024178882A1 WO 2024178882 A1 WO2024178882 A1 WO 2024178882A1 CN 2023103200 W CN2023103200 W CN 2023103200W WO 2024178882 A1 WO2024178882 A1 WO 2024178882A1
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Prior art keywords
amniotic membrane
dry
drying
treatment
preparing
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Chinese (zh)
Inventor
林永亮
罗锦荣
何海娜
吴有陵
刘艳丽
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Geneway Chengdu Biotechnology Co Ltd
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Geneway Chengdu Biotechnology Co Ltd
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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
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

Definitions

  • the invention belongs to the technical field of medical materials, and in particular relates to a preparation method and application of dry amniotic membrane.
  • amnion In the early stages of embryonic development, reptiles, birds, mammals and other animals begin to form a blastocyst membrane around the embryo, and the inner layer of the blastocyst membrane is called the amnion.
  • the amnion is a thin membrane composed of a single layer of epithelial cells connected to each other. It is a translucent membrane that is connected to the amnion layer covering the placenta and umbilical cord.
  • the human amnion is the innermost layer of the human placenta. It contains epithelial cells, is smooth, has no blood vessels, nerves or lymph, has a certain degree of elasticity, and is about 0.02 to 0.5 mm thick.
  • the human amnion has a wide range of clinical applications in wound care, including wound management and postoperative repair in ophthalmology, orthopedics and dentistry.
  • the amnion can prevent leukocyte infiltration and inhibit the activity of multiple proteases. By inhibiting the corresponding proteases, the degree of inflammation is reduced and the duration of inflammation is shortened.
  • the amnion also contains abundant lytic enzymes, lytic bodies and complement, which can inhibit inflammatory responses.
  • Existing amniotic membrane products mainly include wet amniotic membrane and freeze-dried amniotic membrane.
  • the advantage of wet amniotic membrane is that it is softer and can fit the wound surface better during surgery.
  • the disadvantage is that it needs to be stored at low temperature and transported through a cold chain.
  • the advantage of freeze-dried amniotic membrane is that it can be stored at room temperature.
  • the disadvantage is that the material is brittle and is prone to rupture during packaging, storage, transportation and use. It needs to be rehydrated before clinical use, and even after rehydration, it is difficult for the amniotic membrane to return to its original fresh state and it is difficult to completely fit the wound surface.
  • the existing freeze-dried amniotic membrane still has shortcomings in terms of hydrophilicity, promotion of tissue regeneration and resistance to radiation damage.
  • Chinese patent 201610303279.X records a method for preparing an easy-to-preserve biological amniotic membrane.
  • the specification discloses that the processing steps include washing and enzymolyzing the amniotic membrane, filtering, soaking in glycerol, and rinsing with physiological saline to obtain a crude biological amniotic membrane, then taking chitosan and sterile water, mixing and adding phosphoric acid solution, tea polyphenols, casein and ammonium persulfate, mixing and heating again, and adding chitosan to obtain a mixed solution, and then soaking the obtained crude biological amniotic membrane in the mixed solution, through microwave reaction, washing and filtering, and placing in a sterile purification room to dry naturally, thereby obtaining a method for preparing an easy-to-preserve biological amniotic membrane.
  • This method can improve the suture strength of the amniotic membrane to a certain extent, but there is still a need for further improvement in strength,
  • the present invention provides a method for preparing and using dry amniotic membrane, aiming to overcome the shortcomings of the prior art and meet clinical needs to a greater extent; the method for preparing dry amniotic membrane provided by the present invention, the freeze-drying treatment under the protection of an alcohol solution can provide hydrogen bonding, better maintain the flexibility of the natural wet amniotic membrane, and reduce the damage of water crystallization to the structure of the amniotic membrane material during the freeze-drying process.
  • the amniotic membrane prepared by the present invention has enhanced suture strength, which is convenient for surgical suture; increased thickness and hydrophilicity, which is convenient for fitting the wound surface during surgery; stronger performance in promoting tissue regeneration; maintaining the flexibility of the natural wet amniotic membrane and the activity of growth factors and collagen in the amniotic membrane, and can be stored and transported for a long time at room temperature.
  • Toughening treatment The fresh amniotic membrane is subjected to a first step reaction with a compound containing an A-B structural unit to introduce a carbon-carbon double bond structural unit into the fresh amniotic membrane; then the amniotic membrane containing the carbon-carbon double bond structural unit is subjected to a second step reaction with a mixture C of small molecules and biomacromolecules containing carbon-carbon double bond structural units in the presence of an initiator, thereby achieving chemical bonding of the small molecules, biomacromolecules and human amniotic membrane to achieve toughening; wherein the fresh amniotic membrane is human amniotic membrane, as well as the amniotic membrane of pigs and cows.
  • Tissue regeneration promotion treatment The amniotic membrane is immersed in a solution of a substance that promotes tissue regeneration and treated to enhance its tissue regeneration performance;
  • the dried amniotic membrane treated in step (4) is sterilized by ionizing radiation to obtain a dried amniotic membrane product.
  • the ionizing radiation for the irradiation sterilization treatment may be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 20 kGy to 30 kGy and an irradiation time of 18 h to 30 h.
  • a of the A-B structural unit is a primary amine reactive group
  • B is a carbon-carbon double bond structural unit, wherein A is a primary amine reactive group selected from the group consisting of N-hydroxysuccinimide ester, isocyanate and epoxy, and B is acrylic acid or methacrylic acid;
  • the compound containing the A-B structural unit in the optimization scheme is one of N-acryloyloxysuccinimide, methacryloyloxyethyl isocyanate or glycidyl methacrylate.
  • the toughening treatment fresh human amniotic membrane is reacted with a compound containing an A-B structural unit, the mass concentration of the compound containing the A-B structural unit is 1% to 5%, the first step reaction temperature is 4°C to 37°C, and the reaction time is 2h to 48h.
  • the C is a mixture of small molecules and biomacromolecules containing carbon-carbon double bond structural units, wherein the molecular weight of the macromolecule is
  • the molecular weight of small molecules is 10KDa-100KDa, such as gelatin methacrylate; the molecular weight of small molecules is less than 200Da, such as acrylic acid.
  • the reaction temperature of the second step is 4° C. to 37° C.
  • the reaction time is 2 h to 48 h
  • the mass concentration range of small molecules and large molecules is 2% to 10% based on the total amount of C.
  • the added initiator is a mixture of ammonium persulfate and sodium bisulfite, the mass concentration range of the mixture is 0.1% to 1%, and the mass concentration ratio of ammonium sulfate to sodium bisulfite in the optimized solution is 1:1.
  • the initiator can initiate a polymerization reaction between carbon-carbon double bonds.
  • the mass concentration of the hydrophilic macromolecular substance in the thickening and hydrophilic treatment is 10% to 30%, the reaction temperature is 48° C. to 52° C., and the reaction time is 1 h to 3 h; the hydrophilic macromolecular substance includes one or more of gelatin, sodium hyaluronate or chondroitin sulfate.
  • HC-HA/PTX3 complex Heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (PTX3), i.e. heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (PTX3) complex.
  • the mass concentration of HC-HA/PTX3 complex in promoting tissue regeneration is 1% to 5%, the reaction temperature is 3°C to 5°C, and the reaction time is 1h to 3h;
  • the drying method in step (4) includes but is not limited to freeze drying, vacuum drying, air drying or drying with absorbent paper; in the optimized scheme, the irradiated amniotic membrane is gradient dehydrated in an alcohol solution and then dried to obtain a dry human amniotic membrane.
  • the drying process is performed by gradient dehydration in an alcohol solution, wherein the alcohol solution is ethanol and glycerol, and the preferred gradient dehydration method is 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol mixed solution for 2 hours, and 80% ethanol + 20% glycerol mixed solution for 2 hours.
  • the alcohol solution is ethanol and glycerol
  • the preferred gradient dehydration method is 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol mixed solution for 2 hours, and 80% ethanol + 20% glycerol mixed solution for 2 hours.
  • the drying treatment is freeze drying, the freezing temperature is -38°C to -42°C, and the drying time is 48h to 72h.
  • the dry amniotic membrane prepared by the preparation method of the present invention has the following effects:
  • the "mixture of human amniotic membrane containing carbon-carbon double bond structural units and small molecules (molecular weight less than 200Da) and biological macromolecules (molecular weight of 10KDa to 100KDa) containing carbon-carbon double bond structural units" used in the toughening treatment of the present invention small molecules (molecular weight less than 200Da) containing carbon-carbon double bond structural units are easy to penetrate and diffuse in the gaps of the amniotic membrane, and biological macromolecules (molecular weight of 10KDa to 100KDa) containing carbon-carbon double bond structural units can play a role of physical entanglement on the surface of the amniotic membrane.
  • these two substances are used to undergo a co-polymerization chemical reaction with the amniotic membrane containing carbon-carbon double bond structural units, which effectively improves the toughness of the amniotic membrane, that is, the suture strength.
  • this treatment scheme has a greatly improved toughening effect.
  • Freeze-drying under the protection of alcohol solution can provide hydrogen bonding to better maintain the softness of the natural wet amniotic membrane, reduce the damage of water crystallization to the structure of the amniotic membrane material during the freeze-drying process, and maintain the activity of growth factors and collagen in the amniotic membrane.
  • Terminal irradiation sterilization ensures the sterility level and virus inactivation effect of the final prepared dry amniotic membrane, greatly improving the safety of clinical use.
  • the dry amniotic membrane prepared by the method of the present invention can be used to inhibit infection and inflammatory response, reduce scar formation and protect exposed nerve roots in ophthalmic surgery, skin defect repair, tendon repair and adhesion prevention, and spinal surgery.
  • the prepared dry amniotic membrane can be stored and transported for a long time at room temperature.
  • FIG1 is a schematic diagram of the reaction of the preparation method of the present invention.
  • FIG2 is a comparison diagram of suture strength test in the present invention.
  • FIG3 is a comparison diagram of thickness test in the present invention.
  • FIG4 is a comparative diagram of the test of promoting tissue regeneration performance in the present invention.
  • FIG5 is a comparative diagram of the radiation protection effect test in the present invention.
  • FIG. 6 is a comparison diagram of the amniotic membrane in the present invention.
  • the left picture is the amniotic membrane prepared by conventional freeze-drying method as control example 1, which shows great brittleness; the right picture is the amniotic membrane prepared by the preferred embodiment of the present invention, which shows good toughness)
  • Toughening treatment The fresh amniotic membrane is reacted with a compound containing an A-B structural unit in the first step to introduce a carbon-carbon double bond structural unit into the fresh human amniotic membrane; then the human amniotic membrane containing a carbon-carbon double bond structural unit is reacted with a mixture C of small molecules and biomacromolecules containing carbon-carbon double bond structural units in the presence of an initiator in the second step to simultaneously achieve chemical bonding of the small molecules, biomacromolecules and human amniotic membrane to achieve toughening, as shown in FIG1 .
  • A is a primary amine reactive group
  • B is a carbon-carbon double bond structural unit, wherein A is a primary amine reactive group, specifically including N-hydroxysuccinimide ester, isocyanate and epoxy, and B is acrylic acid and methacrylic acid
  • C is a mixture of small molecules and biomacromolecules containing carbon-carbon double bond structural units, wherein the molecular weight of the macromolecule is 10KDa-100KDa, such as Gelatin methacrylate; small molecules have a molecular weight less than 200 Da, such as acrylic acid.
  • the compound containing the AB structural unit is one of N-acryloyloxysuccinimide, methacryloyloxyethyl isocyanate or glycidyl methacrylate, and the structural formulas thereof are as follows:
  • the fresh amniotic membrane is reacted with a compound containing an A-B structural unit in the first step, and the mass concentration of the compound containing the A-B structural unit is 1% to 5%.
  • the small molecules with a molecular weight of less than 200 Da containing carbon-carbon double bond structural units in the toughening treatment include acrylic acid, and its structural formula is as follows:
  • the biomacromolecule containing carbon-carbon double bond structural units in the toughening treatment has a molecular weight of 10KDa to 100KDa, which is gelatin methacrylate, and its structural formula is as follows:
  • the second step reaction is carried out under the condition of participation of an initiator, and the mass concentrations of small molecules and biomacromolecules are both 2% to 10%.
  • a method for preparing a dry amniotic membrane comprises the following steps:
  • the amniotic membrane was immersed in a 1% HC-HA/PTX3 complex aqueous solution at a reaction temperature of 4°C for 2 hours. After this step of treatment, the raw material has the function of promoting regeneration in subsequent clinical use.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, and 80% ethanol + 20% glycerol solution for 2 h at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 48 h to obtain a dry human amniotic membrane.
  • Irradiation sterilization The dry amniotic membrane treated in step (5) is sterilized by ionizing radiation (dose of 25 kGy, irradiation time of 24 h) to obtain a dry amniotic membrane product.
  • a method for preparing dry amniotic membrane comprising the following steps:
  • the human amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 15%, the reaction temperature was 48°C, and the reaction time was 3 hours.
  • the human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 2%, the reaction temperature was 3° C., and the reaction time was 3 h.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, and 80% ethanol + 20% glycerol solution for 2 h at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -38°C and a drying time of 50 h to obtain a dry human amniotic membrane.
  • the ionizing radiation can be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 20 kGy and an irradiation time of 30 h.
  • a method for preparing dry amniotic membrane comprises the following steps:
  • the human amniotic membrane with double bond structural units is immersed in a mixed aqueous solution of acrylic acid and gelatin methacrylate, and then an initiator mixed aqueous solution of ammonium sulfate and sodium bisulfite is added to initiate a polymerization reaction at a reaction temperature of 4° C. and a reaction time of 2 hours to obtain a toughened human amniotic membrane.
  • the human amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 10%, the reaction temperature was 50°C, and the reaction time was 2 hours.
  • the human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 1%, the reaction temperature was 4° C., and the reaction time was 2 h.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, and 80% ethanol + 20% glycerol solution for 2 h at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 48 h to obtain a dry human amniotic membrane.
  • the ionizing radiation can be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 30 kGy and an irradiation time of 18 h.
  • a method for preparing dry amniotic membrane comprises the following steps:
  • the human amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 10%, the reaction temperature was 50°C, and the reaction time was 2 hours.
  • the human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 1%, the reaction temperature was 4° C., and the reaction time was 2 h.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, and 80% ethanol + 20% glycerol solution for 2 h at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -42°C and a drying time of 60 h to obtain a dry human amniotic membrane.
  • the ionizing radiation can be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 28 kGy and an irradiation time of 20 h.
  • a method for preparing dry amniotic membrane comprises the following steps:
  • the human amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 10%, the reaction temperature was 50°C, and the reaction time was 2 hours.
  • the human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 2%, the reaction temperature was 4° C., and the reaction time was 2 h.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, that is, it is immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, 80% ethanol + 20% glycerol solution for 2 hours. Drain after taking out, then freeze-dry, freezing temperature -40°C, drying time for 72 hours, to obtain dry human amniotic membrane.
  • the ionizing radiation can be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 22 kGy and an irradiation time of 27 h.
  • a method for preparing dry amniotic membrane comprises the following steps:
  • aqueous solutions of acrylic acid and gelatin methacrylate (molecular weight 100KDa) with a mass concentration of 20%, then mix and stir the two aqueous solutions to obtain a mixed aqueous solution of acrylic acid and gelatin methacrylate with a mass concentration of 10%.
  • aqueous solutions of ammonium sulfate and sodium bisulfite initiators with a mass concentration of 0.2%, then mix and stir the two aqueous solutions to obtain a mixed aqueous solution of ammonium sulfate and sodium bisulfite with a mass concentration of 0.1%.
  • the human amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 10%, the reaction temperature was 50°C, and the reaction time was 2 hours.
  • the human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 5%, the reaction temperature was 4° C., and the reaction time was 2 h.
  • the human amniotic membrane treated in step (3) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, and 80% ethanol + 20% glycerol solution for 2 h at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 72 h to obtain a dry human amniotic membrane.
  • the ionizing radiation can be gamma ray irradiation or high-energy electron beam irradiation, with a dose of 24 kGy and an irradiation time of 25 h.
  • Example 1 Other structures are as in Example 1, wherein the fresh amniotic membrane is bovine amniotic membrane, wherein the first step reaction temperature in the toughening treatment is 37°C, the reaction time is 6 hours, and the mass concentration of the solution containing the A-B structural unit substance is 3%; the second step reaction in the toughening treatment has a reaction temperature of 37°C and a reaction time of 10 hours, and the mass concentration of small molecules and biological macromolecules, based on the total amount of C, is 6%, and the initiator is a mixed aqueous solution of ammonium sulfate and sodium bisulfite with a mass concentration of 1%.
  • the first step reaction temperature in the toughening treatment is 37°C
  • the reaction time 6 hours
  • the mass concentration of the solution containing the A-B structural unit substance is 3%
  • the second step reaction in the toughening treatment has a reaction temperature of 37°C and a reaction time of 10 hours, and the mass concentration of small molecules and biological macromolecules, based on the
  • the mass concentration of the hydrophilic macromolecular substance in the thickening and hydrophilic treatment is 30%, the reaction temperature is 52° C., and the reaction time is 1 hour; the hydrophilic macromolecular substance is a sodium hyaluronate solution.
  • the mass concentration of HC-HA/PTX3 complex in promoting tissue regeneration treatment was 5%, the reaction temperature was 5° C., and the reaction time was 1 h.
  • the drying method is vacuum drying.
  • Example 1 the fresh amniotic membrane is porcine amniotic membrane, wherein the first step reaction temperature in the toughening treatment is 20°C, the reaction time is 20h, and the mass concentration of the solution containing A-B structural unit substances is 4%; the second step reaction, the reaction temperature is 20°C, the reaction time is 15h, the mass concentration of small molecules and biological macromolecules is 7% based on the total amount of C, and the initiator is a mixed aqueous solution of ammonium sulfate and sodium bisulfite with a mass concentration of 0.5%.
  • the first step reaction temperature in the toughening treatment is 20°C
  • the reaction time is 20h
  • the mass concentration of the solution containing A-B structural unit substances is 4%
  • the second step reaction the reaction temperature is 20°C, the reaction time is 15h, the mass concentration of small molecules and biological macromolecules is 7% based on the total amount of C
  • the initiator is a mixed aqueous solution of ammonium sulfate and sodium
  • the mass concentration of the hydrophilic macromolecular substance in the thickening and hydrophilic treatment is 20%, the reaction temperature is 50° C., and the reaction time is 3 hours; the hydrophilic macromolecular substance is chondroitin sulfate solution.
  • the mass concentration of HC-HA/PTX3 complex in the treatment for promoting tissue regeneration was 3%, the reaction temperature was 4° C., and the reaction time was 1 h.
  • the drying process is air drying or blotting with absorbent paper.
  • Comparative Example 1 is a dry amniotic membrane prepared by a conventional freeze-drying method. The steps are as follows:
  • Fresh human amniotic membrane was freeze-dried at a freezing temperature of -40°C and a drying time of 72 hours to obtain dry human amniotic membrane.
  • Comparative Example 2 is a dry amniotic membrane prepared by the method disclosed in the patent application number 201610303279.X "A method for preparing an easily preserved biological amniotic membrane”. The steps are as follows:
  • a cell scraper was used to remove surface impurities on the amniotic membrane.
  • the amniotic membrane after impurities were then immersed in glycerol and allowed to stand for dehydration overnight at 4°C.
  • the membrane was then filtered and rinsed 5 times with normal saline to obtain a crude biological amniotic membrane for later use.
  • Chitosan and sterile water were then mixed at a solid-liquid ratio of 1:3 and placed in a glass container.
  • a phosphate buffer solution with a pH of 4.5 was added to the glass container in an amount of 30% of the volume of sterile water. The mixture was stirred evenly and allowed to stand for 10 minutes. 50% by mass of chitosan was added to the glass container.
  • Comparative Example 3 is a dry amniotic membrane prepared by the "drying treatment" method. The steps are as follows:
  • Fresh human amniotic membrane was dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, and 80% ethanol + 20% glycerol solution for 2 hours at room temperature (25°C). After being taken out, it was drained and then freeze-dried at a freezing temperature of -40°C and a drying time of 48 hours to obtain dry human amniotic membrane.
  • Comparative Example 4 is a dry amniotic membrane prepared by the "toughening treatment” method containing only one small molecule with a molecular weight less than 200 and the “drying treatment” method. The steps are as follows:
  • the human amniotic membrane toughened in step (1) was dehydrated in an alcohol solution in a gradient manner, that is, immersed in 50% ethanol aqueous solution for 2 h, 75% ethanol aqueous solution for 2 h, 75% ethanol + 25% glycerol solution for 2 h, 80% ethanol + 20% glycerol solution for 2 h, and 100% ethanol + 20% glycerol solution for 2 h. Glycerol solution for 2 hours. Drain after taking out, and then freeze-dry, the freezing temperature is -40°C, the drying time is 48 hours, and dry human amniotic membrane is obtained.
  • Comparative Example 5 is a dry amniotic membrane prepared by the "toughening treatment” method containing only biomacromolecules with a molecular weight of 10 to 100 KDa and the “drying treatment” method. The steps are as follows:
  • the human amniotic membrane toughened in step (1) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, and 80% ethanol + 20% glycerol solution for 2 hours at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 48 hours to obtain a dry human amniotic membrane.
  • Comparative Example 6 is a dry amniotic membrane prepared by the "toughening treatment” and “drying treatment” methods. The steps are as follows:
  • the human amniotic membrane toughened in step (1) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, and 80% ethanol + 20% glycerol solution for 2 hours at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 48 hours to obtain a dry human amniotic membrane.
  • Comparative Example 7 is a dry amniotic membrane prepared by the "thickening and hydrophilic treatment” and “drying treatment” methods. The steps are as follows:
  • Fresh amniotic membrane was immersed in a gelatin aqueous solution with a mass concentration of 10%, the reaction temperature was 50°C, and the reaction time was 2 hours.
  • the human amniotic membrane toughened in step (1) is dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, and 80% ethanol + 20% glycerol solution for 2 hours at room temperature (25°C). After being taken out and drained, it is then freeze-dried at a freezing temperature of -40°C and a drying time of 48 hours to obtain a dry human amniotic membrane.
  • Comparative Example 8 is a dry amniotic membrane prepared by the "promoting tissue regeneration treatment” and “drying treatment” methods. The steps are as follows:
  • Fresh human amniotic membrane was immersed in an aqueous solution of HC-HA/PTX3 complex with a mass concentration of 1%, the reaction temperature was 4° C., and the reaction time was 2 h.
  • the amniotic membrane was dehydrated in an alcohol solution in a gradient manner, i.e., immersed in 50% ethanol aqueous solution for 2 hours, 75% ethanol aqueous solution for 2 hours, 75% ethanol + 25% glycerol solution for 2 hours, and 80% ethanol + 20% glycerol solution for 2 hours at room temperature (25°C). After being taken out, it was drained and then freeze-dried at a freezing temperature of -40°C and a drying time of 48 hours to obtain dry human amniotic membrane.
  • This test includes the following materials: Examples 1-6 and Comparative Examples 1-8.
  • Examples 1-6 are dry amniotic membranes prepared by the five steps of the present invention. It is a dry amniotic membrane prepared by the conventional freeze-drying method.
  • Comparative Example 2 is a dry amniotic membrane prepared by the method disclosed in the patent "A method for preparing an easy-to-preserve biological amniotic membrane” with reference to the application number 201610303279.X.
  • Comparative Example 3 is a dry human amniotic membrane prepared by the "drying treatment” method.
  • Comparative Example 4 is a dry human amniotic membrane prepared by the "toughening treatment” method containing only a small molecule with a molecular weight of less than 200Da and the "drying treatment” method.
  • Comparative Example 5 is a dry human amniotic membrane prepared by the "toughening treatment” method containing only biological macromolecules with a molecular weight of 10 to 100KDa and the "drying treatment” method.
  • Comparative Example 6 is a dry human amniotic membrane prepared by the "toughening treatment” and “drying treatment” methods.
  • Comparative Example 7 is a dry human amniotic membrane prepared by the “thickening and hydrophilic treatment” and “drying treatment” methods.
  • Comparative Example 8 is a dry human amniotic membrane prepared by the "tissue regeneration promotion treatment” and “drying treatment” methods.
  • the suture strength of the dry human amniotic membrane in Example 1-6 is greatly improved, and the "mixture of human amniotic membrane containing carbon-carbon double bond structural units, small molecules with a molecular weight less than 200 containing carbon-carbon double bond structural units, and biological macromolecules with a molecular weight of 10 to 100 KDa" used in the toughening treatment, compared with using only one small molecule containing a carbon-carbon double bond structural unit (Comparative Example 4) or biological macromolecule (Comparative Example 5), its toughening effect is greatly improved.
  • the amniotic membrane of Control Example 1 which is prepared by a conventional freeze-drying method, exhibits greater brittleness; the amniotic membrane prepared by the preferred embodiment of the present invention exhibits good toughness.
  • This test includes the following materials: Examples 1-6 and Comparative Examples 1-8.
  • Examples 1-6 are dried human amniotic membranes prepared by the five steps described in the present invention.
  • Comparative Example 1 is a dried human amniotic membrane prepared by an existing conventional freeze-drying method.
  • Comparative Example 2 is a dried human amniotic membrane prepared by the method disclosed in the patent "A Method for Preparing Easy-to-Preserve Biological Amniotic Membrane" with reference to the application number 201610303279.X.
  • Comparative Example 3 is a dried human amniotic membrane prepared by the "drying treatment” method.
  • Comparative Example 4 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only a small molecule with a molecular weight of less than 200 and the "drying treatment” method.
  • Comparative Example 5 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only biological macromolecules with a molecular weight of 10 to 100 KDa and the "drying treatment” method.
  • Comparative Example 6 is a dried human amniotic membrane prepared by the "toughening treatment” and “drying treatment” methods.
  • Comparative Example 7 is a dried human amniotic membrane prepared by the “thickening and hydrophilic treatment” and “drying treatment” methods.
  • Comparative Example 8 is a dry human amniotic membrane prepared by the "tissue regeneration promotion treatment” and “drying treatment” methods.
  • This test includes the following materials: Examples 1-6 and Comparative Examples 1-8.
  • Examples 1-6 are dried human amniotic membranes prepared by the five steps described in the present invention.
  • Comparative Example 1 is a dried human amniotic membrane prepared by an existing conventional freeze-drying method.
  • Comparative Example 2 is a dried human amniotic membrane prepared by the method disclosed in the patent "A Method for Preparing Easy-to-Preserve Biological Amniotic Membrane" with reference to the application number 201610303279.X.
  • Comparative Example 3 is a dried human amniotic membrane prepared by the "drying treatment” method.
  • Comparative Example 4 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only a small molecule with a molecular weight of less than 200 and the "drying treatment” method.
  • Comparative Example 5 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only biological macromolecules with a molecular weight of 10 to 100 KDa and the "drying treatment” method.
  • Comparative Example 6 is a dried human amniotic membrane prepared by the "toughening treatment” and “drying treatment” methods.
  • Comparative Example 7 is a dried human amniotic membrane prepared by the “thickening and hydrophilic treatment” and “drying treatment” methods.
  • Comparative Example 8 is a dry human amniotic membrane prepared by the "tissue regeneration promotion treatment” and “drying treatment” methods.
  • Example 4 Irradiation Protection Effect Test
  • This test includes the following materials: Examples 1-6 and Comparative Examples 1-8.
  • Examples 1-6 are dried human amniotic membranes prepared by the five steps described in the present invention.
  • Comparative Example 1 is a dried human amniotic membrane prepared by an existing conventional freeze-drying method.
  • Comparative Example 2 is a dried human amniotic membrane prepared by the method disclosed in the patent "A Method for Preparing Easy-to-Preserve Biological Amniotic Membrane" with reference to the application number 201610303279.X.
  • Comparative Example 3 is a dried human amniotic membrane prepared by the "drying treatment” method.
  • Comparative Example 4 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only a small molecule with a molecular weight of less than 200 and the "drying treatment” method.
  • Comparative Example 5 is a dried human amniotic membrane prepared by the "toughening treatment” method containing only biological macromolecules with a molecular weight of 10 to 100 KDa and the "drying treatment” method.
  • Comparative Example 6 is a dried human amniotic membrane prepared by the "toughening treatment” and “drying treatment” methods.
  • Comparative Example 7 is a dried human amniotic membrane prepared by the “thickening and hydrophilic treatment” and “drying treatment” methods.
  • Comparative Example 8 is a dry human amniotic membrane prepared by the "tissue regeneration promotion treatment” and “drying treatment” methods.
  • Postoperative observation Observe wound healing at 1, 2, 3, and 4 weeks after surgery, measure wound size, and calculate wound healing rate. Take healing area tissue for pathological observation 4 weeks after surgery.
  • Wound healing rate healing area/original wound area ⁇ 100
  • the wound healing rate between the two groups was analyzed using t-test, and p ⁇ 0.05 was considered statistically significant.
  • Control group more exudate and varying degrees of necrosis were seen in all patients 1 week after injury, dark red scabs were seen in 2 weeks, and the wound surface was clear in 3 weeks. The wound was significantly reduced in size, the scab became smaller and lighter in color, and the wound healed in 4 weeks.
  • the dry amniotic membrane provided by the present invention has good biocompatibility, no immune rejection, is non-toxic and sterile. Compared with traditional oil gauze, it can accelerate wound tissue healing, reduce scar formation, and can partially regenerate wound skin tissue completely.
  • the experimental group was implanted with the sample prepared in Example 3 of the present invention
  • the control group 1 was a polylactic acid membrane group
  • the control group 2 was a blank control group.
  • Amniotic membrane experimental group 20 New Zealand white rabbits (2 ⁇ 0.5kg) were anesthetized with intravenous injection of sodium pentobarbital. The rabbits were placed in a fixed box and the left limb of the rabbit was extended out of the box. The hair of the left hind limb of the rabbit was removed with a razor, and then disinfected with iodine tincture. The surgical site was the flexor tendon of the rabbit's middle finger. A longitudinal incision was made at the surgical site with a scalpel to find the flexor tendon. After it was lifted with a vascular forceps, a defect was created with surgical scissors (the width was about 1/2 of the tendon). The defective tendon was sutured with a Bunnell suture method.
  • the suture used was a 5-0 single-strand polypropylene suture. After suturing, the biological amniotic membrane prepared by our company was used to wrap the suture. After cleaning and stopping the bleeding, the external wound was sutured with 4-0 silk thread. Finally, the surgery was sealed with plaster. Limb immobilization for 3 weeks.
  • Polylactic acid membrane group 20 New Zealand white rabbits were given the same surgical method as the experimental group. After tendon suture, the suture was wrapped with polylactic acid anti-adhesion membrane, and then the peripheral wound was sutured and fixed with plaster.
  • Blank control group 20 New Zealand white rabbits were given the same surgical method as the experimental group, except that the peripheral wound was sutured directly after the tendon was sutured and fixed with plaster.
  • the effectiveness of amniotic membrane group, polylactic acid membrane group and blank control group were compared by using rabbit toe model.
  • the results showed that the amniotic membrane group showed the best anti-adhesion effect.
  • the effective rate of preventing adhesion of rabbit toe flexor tendon in the amniotic membrane group was 100.00% (20/20)
  • the effective rate of preventing adhesion of rabbit toe flexor tendon in the polylactic acid membrane group was 85.00% (17/20)
  • the effective rate of preventing adhesion of rabbit tendon in the blank control group was 20.00% (4/20).
  • amniotic membrane group was compared with the polylactic acid membrane group and the blank control group, *p ⁇ 0.05;
  • 60 adult New Zealand rabbits weighing 2.0-3.0 kg were selected, regardless of gender. According to the different epidural coverings at the vertebral lamina defect, the 60 rabbits were randomly divided into a dry amniotic membrane group, a polylactic acid membrane group and a blank control group, with 20 rabbits in each group.
  • Specimen collection and processing Five animals were killed in each group at 2, 4, 8, and 12 weeks after surgery. After the animals were killed, the L5 segment of the spine, including the sacrospinal muscles and vertebral attachments, was completely removed, fixed with formalin solution, and then embedded in paraffin and sliced with a thickness of 5 ⁇ m. The tissue sections were stained with HE, Masson, and picric acid-sirius red.
  • the polylactic acid membrane group and the amniotic membrane group had fewer fibroblasts, scarce collagen fibers, and an epidural space.
  • the amniotic membrane was fused with the fibrous tissue behind it, with a fuzzy structure and no adhesion with the dura mater; the collagen density was low, and there was no obvious inflammatory reaction.
  • the granulation tissue was fibrotic, the collagen in the blank group was dense, the dura mater adhered to the scar, and bone tissue was formed at the defect of the original lamina, and there was a scar between it and the dura mater.
  • the polylactic acid membrane group had low collagen density and no obvious inflammatory reaction; there were few epidural scars and no adhesion to the dura mater; the polylactic acid membrane degraded into small red-stained, structureless fragments.
  • a large number of collagen fibers were observed in the blank group, which were arranged tightly and disorderly; the cellular components basically disappeared, and the dura mater was densely adhered to the scar and connected to the new vertebral lamina.
  • the scar area was reduced, the cellular components were scarce, there was no adhesion on the dura mater surface under the new bone plate, some specimens had epidural fat regeneration, and the amniotic membrane was replaced by collagen.
  • the microcomputer image processing system calculated the ratio of the scar area invading the spinal canal to the area of the entire spinal canal. The three sections were measured and the average was taken to calculate the relative scar index in the spinal canal.
  • the repair of the lamina defect was mainly granulation tissue formation, with less collagen fibers, and the scar index difference was not significant (P>0.05).
  • the collagen fibers increased and the scar was initially formed.
  • the value of the amniotic membrane group was the smallest, showing the effect of dry amniotic membrane in preventing adhesion (P ⁇ 0.01).
  • the polylactic acid membrane gradually expanded into a gel, exerting its adhesion prevention properties, but it was still worse than the amniotic membrane group (P ⁇ 0.01).
  • the scar index of the amniotic membrane group and the polylactic acid membrane group was smaller than that of the blank group (P ⁇ 0.01), but the difference between the two groups was significant (P ⁇ 0.05) (see Table 10).
  • the dry amniotic membrane provided by the present invention can protect the exposed nerve roots during spinal surgery by inhibiting infection and inflammatory response, reducing the release of inflammatory mediators, reducing local fibroblast infiltration, reducing scar formation, and preventing adhesion.
  • the wound healing was observed one week after modeling.
  • the models with mild burns that had healed on their own and the models with severe burns of the entire cornea were removed, and moderate and mild burns were selected. 16 models whose self-repair ability could not heal the wound were used for repair experiments.
  • Surgical method All surgical operations were performed using sterile surgical instruments and strictly in accordance with clinical surgical requirements.
  • Eight burn models (2 ⁇ 0.5kg) were randomly selected and anesthetized by intravenous injection of sodium pentobarbital.
  • the front and rear limbs were fixed with ropes.
  • the whole body of the experimental rabbit was wrapped with surgical towels.
  • the hair around the right eye of the rabbit was removed with an electric shaver and disinfected with iodine tincture to minimize the impact on the surgical process and later corneal repair.
  • the surgical site was the wound area of the corneal acid burn model. The damaged area was first debrided under a microscope, and the necrotic corneal epithelium and tissue were thoroughly cleaned with a gem knife.
  • the dry amniotic membrane prepared in Example 6 was taken and soaked in sterile saline, rehydrated for 30-40min, and then the amniotic membrane (epithelial surface facing up) was flatly covered on the entire corneal surface. Next, a layer of contact lenses was covered on the entire cornea (covering the amniotic membrane), and the excess amniotic membrane material was turned up to wrap the edge of the contact lens as protection to prevent the suture from tearing the contact lens.
  • the contact lens was fixed with 10-0 nylon thread in a "rice" shape with 8 stitches. The amniotic membrane material was replaced as appropriate two weeks after surgery. After the fourth week, the amniotic membrane and contact lenses were completely removed.
  • the evaluation index is 0-1 points for effective, 2-4 points for invalid. When evaluating the overall effectiveness, if a single item is invalid, it is judged as invalid. After the evaluation, all data are statistically analyzed.
  • a rabbit corneal acid burn model was used to conduct a comparative study on the effectiveness of the amniotic membrane group and the blank group.
  • the results showed that the dry amniotic membrane group provided by the present invention can better assist in repairing mild corneal acid burns, and the evaluation results 8 weeks after surgery were significantly different from those of the blank group (P ⁇ 0.05).
  • the effective rate of the dry amniotic membrane provided by the present invention in repairing corneal acid burns was 87.5% (7/8), and the effective rate of the blank control group was 25% (2/8).
  • the effective rate of the experimental group (amniotic membrane group) and the blank control group P ⁇ 0.05.

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Abstract

La présente invention appartient au domaine technique des matériaux médicaux, et concerne un procédé de préparation d'une membrane amniotique sèche et son utilisation. Les étapes de préparation comprennent : un traitement de renforcement, un épaississement et un traitement hydrophile, un traitement favorisant la régénération tissulaire, un traitement de séchage et similaires. Selon le procédé de préparation de la membrane amniotique sèche selon la présente invention, un traitement de lyophilisation sous la protection d'une solution alcoolique peut fournir un effet de liaison hydrogène, ce qui permet de mieux maintenir la flexibilité d'une membrane amniotique humide naturelle et de réduire l'endommagement de la structure du matériau de membrane amniotique dans un processus de lyophilisation par cristallisation d'eau. La membrane amniotique préparée par la présente invention présente une résistance à la suture améliorée, facilitant la suture chirurgicale ; possède une épaisseur et un caractère hydrophile accrus, facilitant l'ajustement sur une surface de plaie pendant l'opération ; possède de meilleures performances favorisant la régénération tissulaire ; et possède les caractéristiques de maintien de la flexibilité de la membrane amniotique humide naturelle et de l'activité des facteurs de croissance et du collagène dans la membrane amniotique et similaires, et peut être stockée et transportée à température ambiante pendant une longue durée.
PCT/CN2023/103200 2023-03-02 2023-06-28 Procédé de préparation de membrane amniotique sèche et son utilisation Pending WO2024178882A1 (fr)

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CN116570753B (zh) * 2023-07-13 2023-09-22 健诺维(成都)生物科技有限公司 一种组织再生型生物膜组织复合物及制备方法及应用
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