WO2019109818A1 - 一种干态动物源性胶原纤维组织材料及其制备方法 - Google Patents

一种干态动物源性胶原纤维组织材料及其制备方法 Download PDF

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WO2019109818A1
WO2019109818A1 PCT/CN2018/117165 CN2018117165W WO2019109818A1 WO 2019109818 A1 WO2019109818 A1 WO 2019109818A1 CN 2018117165 W CN2018117165 W CN 2018117165W WO 2019109818 A1 WO2019109818 A1 WO 2019109818A1
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tissue material
preparation
derived collagen
fatty alcohol
collagen fiber
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PCT/CN2018/117165
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English (en)
French (fr)
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张泉娟
赵婧
桂宝珠
陈国明
李�雨
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上海微创心通医疗科技有限公司
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Publication of WO2019109818A1 publication Critical patent/WO2019109818A1/zh

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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
    • A61L27/3604—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
    • A61L27/362—Skin, e.g. dermal papillae
    • 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
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • 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
    • 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
    • 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

Definitions

  • the invention relates to a biological tissue and a preparation method thereof, in particular to a dry animal-derived collagen fiber tissue material and a preparation method thereof.
  • animal-derived collagen fiber tissue materials used in biological tissues commonly used in clinical practice are usually stored using chemical reagents such as glutaraldehyde and/or formaldehyde, or cross-linked with the chemical reagents. deal with.
  • the biological tissue is typically stored in a dilute aqueous solution containing glutaraldehyde and/or formaldehyde to maintain its ingredients in a sterile environment and in a hydrated state.
  • the principle of this method is to freeze the water molecules in the animal-derived tissue to minus 50-80 ° C to form ice crystals, then vacuum and maintain low pressure. Under the conditions, the frozen ice crystals sublime into a gaseous state, the animal source tissue becomes dry, and the glutaraldehyde residue is reduced in the process. (See [2] Mladenov A, Tsvetkov D, Vulchanov L. Freeze drying of biomaterials for the medical Practice. Cryobiology, 1993; 30: 335-48.
  • Patent CN99807736.4 describes a solution for treating tissue by first immersing the tissue in a gradient-increasing polar organic solution (selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, acetone and methyl ethyl ketone) and then immersing it.
  • a gradient-increasing polar organic solution selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, acetone and methyl ethyl ketone
  • aqueous glycerol or low molecular weight ( ⁇ 1000 D) polyethylene glycol
  • polyethylene glycol and heparin solutions of 6000-15000D Thereafter, the tissue was briefly immersed in an aqueous heparin solution for freezing and lyophilization.
  • Patent CN201410096046.8 relates to a surgically implanted biological tissue comprising a polyol (selected from one or a combination of: glycerol, propylene glycol, a derivative of glycerol and a derivative of propylene glycol) and a C1-C3 alcohol (selected from methanol)
  • a non-aqueous treatment solution of ethanol, isopropanol and n-propanol contacts the biological tissue and removes a portion of the treatment solution from the solution-treated biological tissue, the treatment being capable of maintaining a substantially dry state of the tissue.
  • the treated tissue is prone to curling after removing part of the treatment solution, and the residual part of the treatment agent is not easily removed, thereby affecting the sterilization effect, and the simple alcohol dehydration treatment cannot properly control the "basic dry state".
  • the remaining water content in the tissue water content greater than 30% will affect the EO sterilization effect leading to incomplete sterilization).
  • the technical problem to be solved by the invention is to provide a dry animal-derived collagen fiber tissue material and a preparation method thereof, the raw material source is widely and inexpensive, the preparation method is simple and quick, and the prepared dry animal-derived collagen fiber tissue material has good flexibility. It is not easy to curl, has a certain tensile strength, and has good rehydration performance. It is rehydrated quickly in physiological saline.
  • the technical solution adopted by the present invention to solve the above technical problem is to provide a method for preparing a dry animal-derived collagen fiber tissue material, comprising the following steps: S1: cleaning the animal-derived collagen fiber tissue material treated by the crosslinking agent S2: immersing the washed tissue material in a short-chain fatty alcohol having a reduced water content gradient, an aqueous solution of a polyhydric sugar or an aqueous solution of a short-chain fatty alcohol or a polyhydric alcohol; S3: after being immersed in the step S2 The surface residual solution of the tissue material is removed, and then the tissue material is placed in a vacuum dryer, the gradient is increased in vacuum, the moisture in the tissue material is gradually removed, and the short-chain fatty alcohol is removed; S4: removing the tissue material seal After packaging, sterilization is carried out to obtain a dry animal-derived collagen fiber tissue material.
  • the animal-derived collagen fiber tissue material treated by the crosslinking agent in the step S1 is a heterogeneous or allogeneic biological tissue material cross-linked by a crosslinking agent.
  • animal-derived collagen fiber tissue material is a pericardium, a heart valve, a mulch, a pleura, a small intestinal submucosa, a dura mater, a dura mater, a ligament or a skin.
  • the cleaning process in the step S1 is as follows: using physiological saline, a phosphate buffer having a pH of 6.8 to 8.6 or a D-Hanks solution having a pH of 6.8 to 8.6, and washing at room temperature for 3 to 5 times. Every 3 to 5 minutes.
  • the crosslinking agent is one or more of glutaraldehyde, genipin, proanthocyanidins, carbodiimides, and N-hydroxysuccinimide.
  • the short-chain fatty alcohol in the step S2 is a C1-C3 fatty alcohol having a boiling point of not more than 100 ° C and a melting point ranging from -130 ° C to -80 ° C.
  • the short-chain fatty alcohol is one or more of methanol, ethanol, isopropanol and n-propanol.
  • the polyol is a low molecular polyol or/and a polymer having a molecular weight of less than 1000 D using the low molecular polyol as a monomer.
  • polyol is a polyether diol and/or a C2 to C6 polyol.
  • the C2 ⁇ C6 polyol is selected from the group consisting of triethylene glycol, 1,2,6-hexanetriol, 1,2,4-butanetriol, 1,3-butanediol, and 1,4-butylene One of a diol, 1,5-pentanediol, and glycerin.
  • the polysaccharide in the step S2 is a monosaccharide, disaccharide, trisaccharide, polysaccharide or sugar alcohol sugar having water absorption and moisturizing action.
  • polysaccharide is one or more of fructose, sucrose, trehalose, sorbitol, and mannitol.
  • the water content in the aqueous solution is reduced from 70% to 90% to 5% to 15%, and the gradient difference of the water content in the aqueous solution ranges from 5% to 20%.
  • volume ratio of the short-chain fatty alcohol and the polysaccharide in the aqueous solution is (1:3) to (8:1), or the volume ratio of the short-chain fatty alcohol and the polyol is (1:3) to (8). :1).
  • the immersion temperature is 4 to 37 ° C
  • the immersion time is 5 minutes to 48 hours.
  • the degree of vacuum is increased from 2 to 4 KPa to 2 to 3 MPa, and the ratio of the degree of vacuum is 1.2 to 200 times with respect to the pressure gauge.
  • the processing time of the tissue material is from 30 seconds to 30 minutes under each vacuum condition.
  • the surface residual solution of the tissue material after the immersion treatment is sucked off using a medical grade dust-free cloth.
  • the sealed packaging process in the step S4 is as follows: the tissue material is placed in a liquid-free container or packaging bag for packaging in an environment with an absolute humidity of less than 30% or an inert environment.
  • the sterilization method is EO sterilization, electron beam radiation sterilization or gamma ray irradiation sterilization.
  • Another technical solution adopted by the present invention to solve the above technical problems is to provide a dry animal-derived collagen fiber tissue material which is prepared by any of the above preparation methods.
  • the dry animal-derived collagen fiber tissue material provided by the invention and the preparation method thereof have the following advantages: 1. good compliance and good mechanical properties: by adding short-chain fat, The freezing point of the water is lowered, so that the animal source material remains liquid in vacuum drying, does not form ice crystals, and avoids the damage of the collagen fiber structure by vacuuming. At the same time, different concentration gradient dehydration methods and different gradient vacuum drying methods can prevent the gaps and fractures between the fibers formed by the rapid loss of water molecules, and have a better protection effect on the three-dimensional structure of the tissue; The biological tissue material has good compliance, is not easy to curl, and has a certain tensile strength.
  • Example 1 is a HE staining diagram of a dry bovine pericardium treated in Example 1 of the present invention.
  • the invention provides a method for preparing a dry animal-derived collagen fiber tissue material, comprising the following steps:
  • S1 cleaning the animal-derived collagen fiber tissue material treated by the crosslinking agent.
  • the animal-derived collagen fiber tissue material cross-linked by the crosslinking agent is a heterogeneous or allogeneic biological tissue material cross-linked by a crosslinking agent, such as a pericardium, a heart valve, a lamella, a pleura, a small intestine mucosa. Lower layer, dura mater, dura mater, ligament and skin.
  • the crosslinking agent used in the treatment of the animal-derived collagen fibrous tissue material in the present invention is not particularly limited, and examples thereof include commonly used glutaraldehyde, such as Genipin, proanthocyanidins, carbodiimides and N-hydroxyamber.
  • a new type of crosslinking agent such as acid imine or a mixture of the above crosslinking agents.
  • the cleaning solution for cleaning the animal-derived collagen fiber tissue material is physiological saline, or a phosphate buffer solution having a pH of 6.8 to 8.6, or a D-Hanks solution having a pH of 6.8 to 8.6 (ie, Hanks without calcium and magnesium ions). liquid). Preferably, it is washed 3 to 5 times at room temperature for 3 to 5 minutes per pass.
  • the washed tissue material is sequentially immersed in an aqueous solution of a short-chain fatty alcohol having a gradient of concentration, a polysaccharide, or an aqueous solution of a short-chain fatty alcohol or a polyhydric alcohol.
  • the short-chain fatty alcohol is a C1-C3 fatty alcohol having a boiling point of not more than 100 ° C and a melting point of -130 ° C to -80 ° C.
  • the short-chain fatty alcohol is one or more of methanol, ethanol, isopropanol and n-propanol.
  • the polyol is a low molecular polyol and a polymer thereof ( ⁇ 1000 D, ie, the largest polymer molecule in the polymer has a molecular weight of less than 1000 D).
  • the polyol is a polyether diol (having the formula OH-(R-O-)n-R-OH, R is an alkylene group, preferably a C2 to C6 alkylene group) and/or a C2 to C6 polyol.
  • the C2-C6 polyol is preferably triethylene glycol, 1,2,6-hexanetriol, 1,2,4-butanetriol, 1,3-butanediol, 1,4-butanediol, One or more of 1,5-pentanediol and glycerin.
  • the polysaccharide is a monosaccharide, disaccharide, trisaccharide, polysaccharide or sugar alcohol sugar having water absorption and moisturizing action.
  • the polysaccharide is one or more of fructose, sucrose, trehalose, sorbitol, and mannitol.
  • the tissue material is sequentially immersed in a solution of a short-chain fatty alcohol having a gradient of concentration, an aqueous solution of a polysaccharide or an aqueous solution of a short-chain fatty alcohol or a polyhydric alcohol, which means that the tissue material is sequentially immersed until the water content (v/v) is gradually decreased.
  • the aqueous solution of the short-chain fatty alcohol or the polysaccharide or the tissue material is sequentially immersed in an aqueous solution of a short-chain fatty alcohol or a polyhydric alcohol whose water content (v/v) is gradually reduced. As the water content gradually decreases, the content of the polysaccharide or polyol gradually increases.
  • the water content (v/v) gradient of the aqueous solution is reduced and the water content is reduced from 70% to 90% to 5% to 15%.
  • the water content (v/v) gradient difference ranges from 5% to 20%, which may be either an equal difference reduction or a non-equal reduction.
  • the ratio of the short-chain fatty alcohol and the polysaccharide in the present invention is not particularly limited. In a preferred embodiment, the volume ratio of the short-chain fatty alcohol and the polysaccharide is (1:3) to (8:1); The ratio of the chain fatty alcohol and the polyol is not particularly limited. In a preferred embodiment, the volume ratio of the short-chain fatty alcohol to the polyol is (1:3) to (8:1).
  • the tissue material is sequentially immersed in an aqueous solution having a gradient change
  • the immersion temperature is 4 to 37 ° C
  • the immersion time is 5 minutes to 48 hours, preferably 30 minutes to 24 hours. More preferably, it is 1 to 12 hours.
  • step S3 The tissue material processed in step S2 is taken out, the residual solution on the tissue surface is removed, and then placed in a vacuum desiccator, the gradient is lowered to reduce the degree of vacuum, and the moisture in the tissue and the short-chain fatty alcohol are gradually removed.
  • the tissue surface residual solution is aspirated using a medical grade lint-free cloth.
  • the vacuum condition (relative pressure, ie the difference between the standard atmospheric pressure and the absolute pressure) increases in gradient.
  • the degree of vacuum is increased from 2 to 4 KPa to 2 to 3 MPa, and the ratio of the degree of vacuum of the gradient is 1.2 to 200 with respect to the pressure gauge.
  • the ratio of the increased vacuum of the gradient may be a non-equal increase, or an increase in the equal ratio, or a partial increase of 78.
  • the ratio of the degree of vacuum is 1.2 to 10, more preferably 1.2 to 5, to remove low-boiling substances in the tissue material.
  • the ratio of the degree of vacuum is 1.2 to 10, more preferably 1.2 to 5, to remove high-boiling substances in the tissue material.
  • the ratio of the degree of vacuum during the vacuum treatment of the high-boiling substance to the vacuum treatment of the low-boiling substance is 100 to 200 times.
  • the treatment time of the tissue material after removal of the surface residual solution is from 30 seconds to 30 minutes, preferably from 2 minutes to 10 minutes, under each vacuum condition.
  • the sealed package refers to packaging the above-mentioned tissue material and/or its biological prosthesis into a liquid-free container in an environment having an relative humidity of less than 30% or an inert environment such as nitrogen, argon, or the like. Or in the package.
  • the sterilization refers to sterilization by EO sterilization or electron beam radiation sterilization or gamma radiation irradiation.
  • the freezing point of water is lowered, so that the animal source material remains in a liquid state during vacuum drying, and ice crystals are not formed, thereby avoiding the damage of the collagen fiber structure by vacuuming.
  • the water content is reduced in dehydration mode and the vacuum drying method with different gradients can prevent the voids and fractures between the fibers formed by the rapid loss of water molecules, and has a better protection effect on the three-dimensional structure of the tissue.
  • the biological tissue material obtained by the method has good compliance, is not easy to curl, and has a certain tensile strength.
  • Permeable multi-component solutions with different concentration gradients can be used to initially remove water from animal-derived tissues and free glutaraldehyde and formaldehyde molecules in the tissue; further vacuuming removes low-boiling alcohols and water and reduces residual glutaraldehyde And/or a crosslinking agent such as a formaldehyde molecule. These two steps reduce toxicity due to residual crosslinkers such as aldehydes and reduce the risk of tissue calcification.
  • DMSO dimethyl sulfoxide
  • the prepared dry bovine pericardium tissue has a water content of 10.3 ⁇ 1.5%, a rehydration time of 5.97 ⁇ 0.55 minutes, a heat shrinkage temperature of 85.12 ⁇ 1.57°C, a cytotoxicity of 1 grade, and a maximum tensile strength of 12.5 ⁇ . 6.6N.
  • the dried bovine pericardium was subjected to histological (HE) staining experiments. See Figure 1. The results showed that the fiber in the bovine pericardium was relatively uniform, continuous, wavy, and collagen fibers in the pericardium. The structure has not been destroyed. It can be seen that the bovine pericardium treated by the dry treatment method used in the present invention can maintain the original structure and state of the tissue.
  • a porcine aortic valve was obtained at a local slaughterhouse and washed with 0.625 wt% glutaraldehyde (Sigma-Aldrich) The solution of Co.LLC.) was fixed for more than 3 days.
  • the fixed porcine aortic valve was washed with physiological saline at room temperature, washed 3 to 5 times, and washed for 3 to 5 minutes each time.
  • the porcine aortic valve was sequentially immersed in No. 1 solution, No. 2 solution, 3 In the No. 4 solution, No. 4 solution, No. 5 solution, and No. 6 solution, the experimental bottle in which the porcine aortic valve was placed was stored in the dark, and immersed at 20 ° C for 30 minutes.
  • the treated tissue material was placed in a medical dust-free cloth to absorb the remaining solution on the surface, and then placed in a vacuum desiccator, and the degree of vacuum was gradually lowered with reference to Table 4.
  • the dry porcine aortic valve is placed in a dialysis bag in a dry, nitrogen-containing environment, sealed, and EO sterilized.
  • the prepared dry porcine aortic valve tissue has a water content of 12.8 ⁇ 1.7%, a rehydration time of 4.88 ⁇ 0.32 minutes, a heat shrinkage temperature of 80.78 ⁇ 1.38°C, a cytotoxicity of grade 1, and a maximum tensile strength of 13.5 ⁇ 3.2N.
  • the submucosal tissue of the small intestine of the pig was obtained at a local slaughterhouse, and after peeling, trimming and washing, it was fixed with carbodiimide.
  • the porcine intestinal mucosa and the cross-linking solution were mixed at a ratio of 8:1 (W/V); at 20 ° C, the mixture was vortexed at 400 r/min for 16 h; then vortexed and washed 8 times for 10 min each time.
  • the fixed subcutaneous tissue of the small intestine of the pig is cut into small pieces (30 mm x 50 mm), and then the submucosal tissue of the fixed small intestine of the pig is washed with physiological saline at room temperature, washed 3 to 5 times, and washed for 3 to 5 minutes each time.
  • the porcine small intestine submucosal tissue was sequentially immersed in the No. 1 solution, the No. 2 solution, the No. 3 solution, the No. 4 solution, the No. 5 solution, and the No. 6 solution, and the experimental bottle in which the porcine small intestine submucosal tissue was placed was protected from light. Immerse for 30 minutes at 20 °C.
  • the treated tissue material was placed in a medical dust-free cloth to absorb the remaining solution on the surface, and then placed in a vacuum desiccator, and the degree of vacuum was gradually lowered with reference to Table 6. Finally, in a dry nitrogen-containing environment, the dry porcine small intestine submucosal tissue is placed in a dialysis bag, sealed, and EO sterilized.
  • the prepared dry porcine small intestine submucosal tissue has a water content of 11.9 ⁇ 1.6%, a rehydration time of 5.66 ⁇ 0.68 minutes, a thermal shrinkage temperature of 82.46 ⁇ 1.38°C, a cytotoxicity of grade 1, and a maximum tensile strength of 11.7 ⁇ 2.8 N.
  • the material is obtained by obtaining the pig pericardium tissue at the local slaughterhouse after being subjected to fat stripping, trimming and washing, and fixing with N-hydroxysuccinimide.
  • the concentration was 0.1 mol/L, and the pig pericardium and cross-linking solution were mixed at a ratio of 8:1 (W/V); at 20 ° C, the mixture was shaken at 400 r/min for 16 h; then vortexed and washed 8 times for 10 min each time. .
  • the fixed pig pericardium was cut into small pieces (30 mm ⁇ 50 mm), and then the fixed pig pericardium was washed with physiological saline at room temperature, washed 3 to 5 times, and washed for 3 to 5 minutes each time.
  • the pig pericardium was sequentially immersed in No. 1 solution, No. 2 solution, No. 3 solution, No. 4 solution, No. 5 solution, No. 6 solution, and the experimental bottle placed in the pig pericardium was stored in the dark and immersed for 30 minutes at 20 ° C. .
  • the treated tissue material was placed in a medical dust-free cloth to absorb the remaining solution on the surface, and then placed in a vacuum desiccator, and the degree of vacuum was gradually lowered with reference to Table 6. Finally, in a dry nitrogen-containing environment, the dry pig pericardium is placed in a dialysis bag, sealed, and EO sterilized.
  • the prepared dry pig pericardium tissue has a water content of 13.9 ⁇ 2.1%, a rehydration time of 5.07 ⁇ 0.35 minutes, a heat shrinkage temperature of 84.64 ⁇ 2.21°C, a cytotoxicity of 1 grade, and a maximum tensile strength of 11.5 ⁇ . 1.8N.
  • the method for preparing the dry animal-derived collagen fiber tissue material has the advantages of wide and low raw material source, simple and rapid preparation method and convenient use.
  • the prepared dry animal-derived collagen fiber tissue material has good flexibility, is not easy to curl, has certain tensile strength, has no residual toxic reagents, and reduces the risk of tissue calcification, and the tissue water content is controlled at 5%-25% (V). /V) between to facilitate subsequent sterilization, and rehydration performance is good, in the clinical operation, the rehydration speed in the physiological saline is fast, generally the original hydration state can be restored in about 5 minutes.

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Abstract

一种干态动物源性胶原纤维组织材料及其制备方法。所述制备方法包括如下步骤：清洗经过交联剂处理后的动物源性胶原纤维组织材料；将清洗后的组织材料依次浸没在浓度呈梯度变化的短链脂肪醇、多元糖的水溶液或者短链脂肪醇、多元醇的水溶液中；将浸没处理后的组织材料表面溶液吸除后放置于真空干燥器中，梯度降低真空度，逐渐去除组织材料中的水分以及短链脂肪醇；取出所述组织材料密封包装后灭菌，即得到干态动物源胶原纤维组织材料。该干态动物源性胶原纤维组织材料柔韧性好、不易卷曲，具有一定的拉伸强度，无残留有毒试剂，降低组织钙化风险，再水合性能好。

Description

一种干态动物源性胶原纤维组织材料及其制备方法 技术领域
本发明涉及一种生物组织及其制备方法，尤其涉及一种干态动物源性胶原纤维组织材料及其制备方法。
 背景技术
当前，临床上常用的生物组织，如生物瓣膜、生物补片等所用的动物源性胶原纤维组织材料通常使用化学试剂如戊二醛和/或甲醛贮存，或用所述化学试剂进行交联固定处理。生物组织一般保存在包含戊二醛和/或甲醛的稀释水溶液中，使其成分保持无菌环境和水合状态。然而，大量的研究证明瓣膜组织在植入后残留的戊二醛会促进瓣膜的钙化，而且戊二醛具有较高的毒性，即使很低的残留量也会在人体产生毒性，不利于内皮化的形成。（参见[1] Mirzaie M, Brunner E, Mahbub-ul Latif AH, et al. A new storage solution for porcine aortic valves. Ann Thorac Cardiovasc Surg, 2007, 13: 102–109.）因此，用戊二醛处理和保存的生物组织假体在植入前必须经过多次大量的清洗以去除戊二醛。但是，在植入生物组织材料或其制备的生物假体时，手术前医疗人员应当尽量减少医疗器械暴露在外的准备工作，因为使用“易得即用”的组织和假体不仅可以降低染菌或误差几率，也可以减少植入时间。所以，开发一种干态生物组织材料的制备方法不仅有广阔的发展前景也具有重要的实际应用价值。
巴西圣保罗大学Pitombo课题组专注于研究生物组织低温冷冻干燥处理方法，该方法的原理是将动物源组织中的水分子冷冻至零下50-80℃，使之形成冰晶，然后再抽真空，保持低压条件下，冷冻的冰晶升华成气态，动物源组织变得干燥，同时在该过程中减少戊二醛残留。（参见[2] Mladenov A, Tsvetkov D, Vulchanov L. Freeze drying of biomaterials for the medical practice. Cryobiology, 1993; 30: 335-48.[3] Camila B, Marina M, Adolfo L, Bronislaw P, Marisa B, Olga H, Ronaldo P. Effect of freeze-drying on the mechanical, physical and morphological properties of glutaraldehyde-treated bovine pericardium: evaluation of freeze-dried treated bovine pericardium properties. J Appl Biomater Biomech, 2010; 8: 186-190.）但是，该处理工艺有以下的缺点：1）力学性能差，在冷冻的过程中，冰的密度相较于水的密度大，当水冷冻为冰晶后，体积变大，会破坏胶原纤维组织结构，所以，这种冻干的组织柔韧性较差，不能防止断裂；2）再水合性能差，这种干态的组织使用前的再水合性能较差，通常需要几天的时间才能恢复至原有的水合状态；3）工艺复杂，耗时且昂贵。
专利CN99807736.4描述了一种溶液处理组织的方法，首先将组织浸没在梯度增加的极性有机溶液（选自甲醇、乙醇、异丙醇、乙腈、丙酮和甲乙酮组成的组中），然后浸没在甘油水溶液或低分子量(<1000D)聚乙二醇中以及6000-15000D的聚乙二醇和肝素溶液中。此后，组织被简短浸没在肝素水溶液中冷冻和冻干。这种脱水过程显著减少组织的总尺寸，且使用的化学试剂（如乙腈、丙酮等）有一定的毒性，此外，该脱水过程的生物组织不能成功地再水合并恢复其原始尺寸。
专利CN201410096046.8涉及一种外科植入的生物组织，用包含多元醇（选自以下之一或组合：甘油、丙二醇、甘油的衍生物和丙二醇的衍生物）和C1-C3醇（选自甲醇、乙醇、异丙醇和正丙醇）的非水性处理溶液接触所述生物组织和从溶液处理过的生物组织去除部分处理溶液，这种处理方法能够组织保持基本干燥状态。但是这种处理后的组织在去除部分处理溶液后易出现卷曲和残留部分处理试剂不易去除而影响灭菌效果，而且采用的单纯的醇类脱水处理并不能较好的控制“基本干燥状态”的组织中的剩余含水量（含水量大于30%会影响EO灭菌效果导致灭菌不彻底）。
总之，现有的生物组织贮存方法如醛类溶液保存和冷冻干燥保存由于存在力学性能差、再水合性能差、工艺复杂且成本高等缺点，在使用上有待改变和提高；而出现的甘油类组织干态脱水方法具有一定优势，但其在生物组织形态变化、残留试剂和组织含水量不能很好地控制等方面仍需要进一步改进。
 技术问题
本发明要解决的技术问题是提供一种干态动物源性胶原纤维组织材料及其制备方法，原料来源广泛低廉，制备方法简单快捷，所制备的干态动物源性胶原纤维组织材料柔韧性好、不易卷曲，具有一定的拉伸强度，再水合性能好，在生理盐水中再水化速度快。
 技术解决方案
本发明为解决上述技术问题而采用的技术方案是提供一种干态动物源性胶原纤维组织材料的制备方法，包括如下步骤：S1：清洗经过交联剂处理后的动物源性胶原纤维组织材料；S2：将清洗后的组织材料依次浸没在水含量梯度降低的短链脂肪醇、多元糖的水溶液或者短链脂肪醇、多元醇的水溶液中；S3：将经过所述步骤S2浸没处理后的组织材料的表面残留溶液移除，然后将所述组织材料放置于真空干燥器中，梯度提高真空度，逐渐去除所述组织材料中的水分以及短链脂肪醇；S4：取出所述组织材料密封包装后进行灭菌，即得到干态动物源性胶原纤维组织材料。
进一步地，所述步骤S1中经过交联剂处理后的动物源性胶原纤维组织材料为交联剂交联处理后的异种或同种异体生物组织材料。
进一步地，所述动物源性胶原纤维组织材料为心包、心脏瓣膜、覆膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带或皮肤。
进一步地，所述步骤S1中的清洗过程如下：用生理盐水、pH值为6.8～8.6的磷酸盐缓冲液或pH值为6.8～8.6的D-Hanks溶液，在室温下清洗3～5遍，每遍3～5分钟。
进一步地，所述交联剂为戊二醛、京尼平、原花青素、碳化二亚胺和N-羟基琥珀酸亚胺中的一种或多种。
进一步地，所述步骤S2中的短链脂肪醇为沸点不大于100℃，熔点范围为-130℃~-80℃的C1-C3脂肪醇。
进一步地，所述短链脂肪醇为甲醇、乙醇、异丙醇和正丙醇中的一种或多种。
进一步地，所述步骤S2中多元醇为低分子多元醇或/和以所述低分子多元醇为单体的分子量小于1000D的-聚合物。
进一步地，所述多元醇为聚醚二醇和/或C2~C6多元醇。
进一步地，所述C2~C6多元醇选自三乙二醇、1,2,6-己三醇、1,2,4-丁三醇、1,3-丁二醇、1,4-丁二醇、1,5-戊二醇和甘油中的一种。
进一步地，所述步骤S2中的多元糖为具有吸水和保湿作用的单糖、双糖、三糖、多糖或糖醇类糖。
进一步地，所述多元糖为果糖、蔗糖、海藻糖、山梨糖醇和甘露糖醇中的一种或多种。
进一步地，所述水溶液中水含量从70%～90%降低至5%～15%，水溶液中水含量的梯度差值范围为5%~20%。
进一步地，所述水溶液中短链脂肪醇、多元糖的体积比例为（1：3）~（8：1），或者短链脂肪醇、多元醇的体积比例为（1：3）~（8：1）。
进一步地，所述步骤S2中的组织材料依次浸没在在所述水溶液时，浸没温度为4~37℃，每次浸没时间为5分钟~48小时。
进一步地，所述步骤S3中真空度从2~4KPa增加到2~3Mpa，相对压强计，所述真空度的比值范围为1.2~200倍。
进一步地，在每个真空度条件下，所述组织材料的处理时间为30秒~30分钟。
进一步地，所述步骤S3中使用医疗级无尘布将浸泡处理后的所述组织材料的表面残留溶液吸除。
进一步地，所述步骤S4中的密封包装过程如下：在相对湿度小于30%的环境或惰性环境将所述组织材料置入无液体的容器或包装袋进行包装。
进一步地，所述的灭菌方式为EO灭菌、电子束辐射灭菌或伽马射线辐照灭菌。
本发明为解决上述技术问题而采用的另一技术方案是提供一种干态动物源性胶原纤维组织材料，所述组织材料通过上述任一制备方法制备得到。
 有益效果
本发明对比现有技术有如下的有益效果：本发明提供的干态动物源性胶原纤维组织材料及其制备方法，具有以下优点：1、顺应性好、力学性能好：通过加入短链脂肪，降低了水的凝固点，使得动物源材料在真空干燥时保持液态，不会形成冰晶，避免了抽真空对胶原纤维结构的破坏。同时，不同浓度梯度的脱水方式以及不同梯度的真空干燥方式，可以防止由于水分子的快速流失而形成的纤维之间的空隙和断裂，对组织的三维结构有较好的保护作用；该方法得到的生物组织材料顺应性好、不易卷曲、具有一定的拉伸强度。2、生物相容性好、生物安全性高：采用不同浓度梯度的渗透性的多组分溶液可以初步去除动物源组织中的水分以及动物源组织中游离的戊二醛和甲醛分子，抽真空可以快速去除低沸点的醇和水以及减少残留的戊二醛和/或甲醛分子，这两个步骤降低了由于醛类残留引起的毒性，并且降低了动物源组织钙化的风险；3、实用性强：原料来源广泛低廉，制备方法简单快捷，使用方便。临床手术操作时，在生理盐水中再水化速度快，一般5分钟左右即可恢复原先水合状态。
 附图说明
图1为本发明专利实施例1中处理后的干态牛心包HE染色图。
 本发明的实施方式
下面结合附图表和实施例对本发明作进一步的描述。
本发明提供的一种干态动物源性胶原纤维组织材料的制备方法，包括以下步骤：
S1：清洗经过交联剂处理后的动物源性胶原纤维组织材料。
所述的经过交联剂交联处理后的动物源性胶原纤维组织材料为交联剂交联处理后的异种或同种异体生物组织材料，例如心包、心脏瓣膜、覆膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带和皮肤。本发明中对处理动物源性胶原纤维组织材料所采用的交联剂没有特别的限制，例如常用的戊二醛，又例如京尼平(Genipin)，原花青素，碳化二亚胺和N-羟基琥珀酸亚胺等新型交联剂，或者以上交联剂的混合。
所述清洗动物源性胶原纤维组织材料的清洗液为生理盐水、或pH值为6.8～8.6的磷酸盐缓冲液，或pH值为6.8～8.6的D-Hanks溶液(即无钙镁离子的Hanks液)。优选，在室温下清洗3～5遍，每遍3～5分钟。
S2:将清洗后的组织材料依次浸没在浓度呈梯度变化的短链脂肪醇、多元糖的水溶液或者短链脂肪醇、多元醇的水溶液中。
所述短链脂肪醇为沸点不大于100℃，熔点为-130℃~-80℃的C1-C3脂肪醇。优选地，所述短链脂肪醇为甲醇、乙醇、异丙醇和正丙醇中的一种或多种。
所述的多元醇为低分子多元醇及以其为单体的聚合物（＜1000D，即聚合物中最大的聚合物分子的分子量小于1000D）。例如，所述多元醇为聚醚二醇( 结构式为 OH-(R-O-)n-R-OH，R 为亚烷基，优选为 C2 ～ C6 亚烷基 )和/或C2~C6多元醇。所述C2~C6多元醇优选为三乙二醇、1,2,6-己三醇、1,2,4-丁三醇、1,3-丁二醇、1,4-丁二醇、1,5-戊二醇和甘油中的一种或多种。
所述的多元糖是具有吸水和保湿作用的单糖、双糖、三糖、多糖或糖醇类糖。例如，所述多元糖为果糖、蔗糖、海藻糖、山梨糖醇和甘露糖醇中的一种或多种。
所述的组织材料依次浸没在浓度呈梯度变化的短链脂肪醇、多元糖的水溶液或者短链脂肪醇、多元醇的水溶液中是指依次将组织材料浸入到水含量（v/v）逐渐减少的短链脂肪醇、多元糖的水溶液中或者依次将组织材料浸入到水含量（v/v）逐渐减少的短链脂肪醇、多元醇的水溶液中。在水含量逐渐减少的同时，多元糖或者多元醇的含量逐渐增加。在一个或多个实施例中，所述的水溶液的水含量（v/v）梯度降低，水含量从70%～90%降低至5%～15%。在一个或多个实施例中，水含量（v/v）梯度差值的范围为5%~20%，可以为等差降低，也可以为非等差降低。本发明对短链脂肪醇、多元糖的比例没有特别的限制，在一个优选的实施例中短链脂肪醇、多元糖的体积比例为（1：3）~（8：1）；同样对短链脂肪醇、多元醇的比例没有特别的限制，在一个优选的实施例中短链脂肪醇、多元醇的体积比例为（1：3）~（8：1）。
此外，在一些实施例中，所述的组织材料依次浸没在浓度呈梯度变化的水溶液时，浸没温度为4～37℃，每次浸没时间为5分钟～48小时，优选30分钟～24小时，更优选地1～12小时。
S3:将步骤S2处理的组织材料取出，将组织表面残余溶液移除后放置于真空干燥器中，梯度降低真空度，逐渐去除组织中的水分以及短链脂肪醇。
在本发明的一些实施例中，使用医疗级无尘布将组织表面残余溶液吸除。所述的真空度条件（相对压强，即标准大气压与绝对压强的差值）梯度增加。在一个实施例中，真空度从2~4KPa增加到2~3Mpa，相对压强计，梯度增加的真空度的比值范围为1.2~200。梯度增加的真空度的比值可以为非等比增加，也可以为等比增加，或者部分等比增加78。优选，低沸点的物质真空处理过程中（例如前1~3次真空处理），真空度的比值为1.2~10，更优选为1.2~5，以去除组织材料中低沸点的物质。后续的高沸点物质真空处理过程中（例如第3~6次真空处理），真空度的比值为1.2~10，更优选为1.2~5，以去除组织材料中高沸点物质。高沸点物质真空处理过程中真空度与低沸点物质真空处理过程中真空度的比值为100~200倍。在一些实施例中，在每个真空度条件下，表面残余溶液移除后的组织材料的处理时间为30秒～30分钟，优选2分钟～10分钟。
S4:取出组织材料密封包装后进行灭菌。
在本发明的一些实施例中，所述的密封包装是指在相对湿度小于30%的环境或惰性环境如氮气、氩气等将上述组织材料和/或其生物假体包装到无液体的容器或包装中。在本发明的一些实施例中，所述的灭菌是指采用EO灭菌或电子束辐射灭菌或伽马射线辐照灭菌。
本发明中，首先通过加入短链脂肪醇，降低了水的凝固点，使得动物源材料在真空干燥时保持液态，不会形成冰晶，避免了抽真空对胶原纤维结构的破坏。其次水含量呈梯度降低的脱水方式以及不同梯度的真空干燥方式，可以防止由于水分子的快速流失而形成的纤维之间的空隙和断裂，对组织的三维结构有较好的保护作用。该方法得到的生物组织材料顺应性好、不易卷曲、具有一定的拉伸强度。第三，两次脱水。采用不同浓度梯度的渗透性的多组分溶液可以初步去除动物源组织中的水分以及组织中游离的戊二醛和甲醛分子；进一步抽真空可以去除低沸点的醇和水以及减少残留的戊二醛和/或甲醛分子等交联剂。这两个步骤降低了由于醛类等交联剂残留引起的毒性，并且降低了组织钙化的风险。
 
实施例 1
在当地屠宰厂获取牛心包组织，经过脂肪剥离、修剪和清洗后用0.625wt%京尼平溶液，即0．9375g京尼平用二甲基亚砜(DMSO)10mL溶解，加PBS(pH=7．4)至150mL，固定3天以上，PH=7.4，将固定后的牛心包裁剪成小片（15mm×15mm），用生理盐水（华仁药业股份有限公司）在室温下清洗牛心包组织小片，清洗3～5次，每次清洗3～5分钟。参照表1将牛心包依次浸没在1号溶液、2号溶液、3号溶液、4号溶液、5号溶液、6号溶液中，放置心包的实验瓶避光保存，20℃下浸没30分钟。之后，将处理后的组织材料放在医用无尘布内吸除表面剩余溶液，随后放置于真空干燥器中，参照表2逐渐降低真空度。最后，在干燥的洁净环境中，将干态的牛心包放入透析袋（Tyvek®，杜邦中国集团有限公司）中，密封，EO灭菌。
Figure 898434dest_path_image001
所制备的干态牛心包组织的含水量为10.3±1.5%，再水化时间为5.97±0.55分钟，热皱缩温度为85.12±1.57℃，细胞毒性为1级，最大拉伸强度为12.5±6.6N。将经过干态处理后灭菌的牛心包使用组织学（HE）染色实验，请参见图1，结果显示牛心包组织中的纤维走向相对一致，连续性好，呈波浪状，心包中的胶原纤维结构没有被破坏。由此可见，本发明使用的干态处理方法处理后的牛心包能够保持组织原先结构和状态。
 
实施例 2
在当地屠宰厂获取猪主动脉瓣膜，经过清洗后用0.625wt%戊二醛（Sigma-Aldrich Co.LLC.）溶液固定3天以上。将固定后的猪主动脉瓣膜用生理盐水在室温下清洗，清洗3～5次，每次清洗3～5分钟，参照表3将猪主动脉瓣膜依次浸没在1号溶液、2号溶液、3号溶液、4号溶液、5号溶液、6号溶液中，放置猪主动脉瓣膜的实验瓶避光保存，20℃下浸没30分钟。之后，将处理后的组织材料放在医用无尘布内吸除表面剩余溶液，随后放置于真空干燥器中，参照表4逐渐降低真空度。最后，在干燥的含氮气的环境中，将干态的猪主动脉瓣膜放入透析袋中，密封，EO灭菌。
Figure 346733dest_path_image002
所制备的干态猪主动脉瓣膜组织的含水量为12.8±1.7%，再水化时间为4.88±0.32分钟，热皱缩温度为80.78±1.38℃，细胞毒性为1级，最大拉伸强度为13.5±3.2N。
 
实施例 3
在当地屠宰厂获取猪小肠粘膜下层组织，经过剥离、修剪和清洗后用碳化二亚胺固定，具体地，用pH=5.5的PBS配制交联液，碳化二亚胺的浓度为0.1mol/L，猪小肠粘膜与交联液以8∶1（W/V）比例混合；于20℃，以400r/min震荡交联16h；然后震荡清洗8次，每次10min。将固定后的猪小肠粘膜下层组织裁剪成小片（30mmx50mm），之后将固定后的猪小肠粘膜下层组织用生理盐水在室温下清洗，清洗3～5次，每次清洗3～5分钟。参照表5将猪小肠粘膜下层组织依次浸没在1号溶液、2号溶液、3号溶液、4号溶液、5号溶液、6号溶液中，放置猪小肠粘膜下层组织的实验瓶避光保存，20℃下浸没30分钟。之后，将处理后的组织材料放在医用无尘布内吸除表面剩余溶液，随后放置于真空干燥器中，参照表6逐渐降低真空度。最后，在干燥的含氮气的环境中，将干态的猪小肠粘膜下层组织放入透析袋中，密封，EO灭菌。
Figure 939519dest_path_image003
所制备的干态猪小肠粘膜下层组织的含水量为11.9±1.6%，再水化时间为5.66±0.68分钟，热皱缩温度为82.46±1.38℃，细胞毒性为1级，最大拉伸强度为11.7±2.8N。
 
实施例 4
材料为在当地屠宰厂获取猪心包组织经过脂肪剥离、修剪和清洗后用N-羟基琥珀酸亚胺固定，具体地，用pH=5.5的PBS配制交联液，N-羟基琥珀酸亚胺的浓度为0.1mol/L，猪心包与交联液以8∶1（W/V）比例混合；于20℃，以400r/min震荡交联16h；然后震荡清洗8次，每次10min。。将固定后的猪心包裁剪成小片（30mm×50mm），之后将固定后的猪心包用生理盐水在室温下清洗，清洗3～5次，每次清洗3～5分钟。参照表7将猪心包依次浸没在1号溶液、2号溶液、3号溶液、4号溶液、5号溶液、6号溶液中，放置猪心包的实验瓶避光保存，20℃下浸没30分钟。之后，将处理后的组织材料放在医用无尘布内吸除表面剩余溶液，随后放置于真空干燥器中，参照表6逐渐降低真空度。最后，在干燥的含氮气的环境中，将干态的猪心包放入透析袋中，密封，EO灭菌。
Figure 865887dest_path_image004
所制备的干态猪心包组织的含水量为13.9±2.1%，再水化时间为5.07±0.35分钟，热皱缩温度为84.64±2.21℃，细胞毒性为1级，最大拉伸强度为11.5±1.8N。
综上所述，本发明提供的干态动物源性胶原纤维组织材料的制备方法，原料来源广泛低廉，制备方法简单快捷，使用方便。所制备的干态动物源性胶原纤维组织材料柔韧性好、不易卷曲，具有一定的拉伸强度，无残留有毒试剂，降低了组织钙化的风险，组织含水量控制在5%-25%（V/V）之间以利于后续灭菌，且再水合性能好，临床手术操作时，在生理盐水中再水化速度快，一般5分钟左右即可恢复原先水合状态。
虽然本发明已以较佳实施例揭示如上，然其并非用以限定本发明，任何本领域技术人员，在不脱离本发明原理的前提下，当可作些许的修改和完善，因此本发明的保护范围当以权利要求书所界定的为准。

Claims (21)

  1. 一种干态动物源性胶原纤维组织材料的制备方法，包括如下步骤：
    S1：清洗经过交联剂处理后的动物源性胶原纤维组织材料；
    S2：将清洗后的组织材料依次浸没在水含量梯度降低的短链脂肪醇、多元糖的水溶液或者短链脂肪醇、多元醇的水溶液中；
    S3：将经过所述步骤S2浸没处理后的组织材料的表面残留溶液移除，然后将所述组织材料放置于真空干燥器中，梯度提高真空度，逐渐去除所述组织材料中的水分以及短链脂肪醇；
    S4：取出所述组织材料密封包装后进行灭菌，即得到干态动物源性胶原纤维组织材料。
  2. 如权利要求1所述的制备方法，其特征在于，所述步骤S1中经过交联剂处理后的动物源性胶原纤维组织材料为交联剂处理后的异种或同种异体生物组织材料。
  3. 如权利要求2所述的制备方法，其特征在于，所述动物源性胶原纤维组织材料为心包、心脏瓣膜、覆膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带或皮肤。
  4. 如权利要求1所述的制备方法，其特征在于，所述步骤S1中的清洗过程如下：用生理盐水、pH值为6.8～8.6的磷酸盐缓冲液或pH值为6.8～8.6的D-Hanks溶液，在室温下清洗3~5遍，每遍3~5分钟。
  5. 如权利要求1所述的制备方法，其特征在于，所述交联剂为戊二醛、京尼平、原花青素、碳化二亚胺和N-羟基琥珀酸亚胺中的一种或多种。
  6. 如权利要求1所述的制备方法，其特征在于，所述步骤S2中的短链脂肪醇为沸点不大于100℃，熔点为-130℃~-80℃的C1-C3脂肪醇。
  7. 如权利要求6所述的制备方法，其特征在于，所述短链脂肪醇为甲醇、乙醇、异丙醇和正丙醇中的一种或多种。
  8. 如权利要求1所述的制备方法，其特征在于，所述步骤S2中多元醇为低分子多元醇或/和以所述低分子多元醇为单体的分子量小于1000D的聚合物。
  9. 如权利要求8所述的制备方法，其特征在于，所述多元醇为聚醚二醇和/或C2~C6多元醇。
  10. 如权利要求9所述的制备方法，其特征在于，所述C2~C6多元醇选自三乙二醇、1,2,6-己三醇、1,2,4-丁三醇、1,3-丁二醇、1,4-丁二醇、1,5-戊二醇和甘油中的一种。
  11. 如权利要求1所述的制备方法，其特征在于，所述步骤S2中的多元糖为具有吸水和保湿作用的单糖、双糖、三糖、多糖或糖醇类糖。
  12. 如权利要求11所述的制备方法，其特征在于，所述多元糖为果糖、蔗糖、海藻糖、山梨糖醇和甘露糖醇中的一种或多种。
  13. 如权利要求1所述的制备方法，其特征在于，所述水溶液中水含量从70%～90%降低至5%～15%，水溶液中水含量的梯度差值范围为5%~20%。
  14. 如权利要求1所述的制备方法，其特征在于，所述水溶液中短链脂肪醇、多元糖的体积比例为（1：3）~（8：1），或者短链脂肪醇、多元醇的体积比例为（1：3）~（8：1）。
  15. 如权利要求1所述的制备方法，其特征在于，所述步骤S2中的组织材料依次浸没在所述水溶液时，浸没温度为4~37℃，每次浸没时间为5分钟~48小时。
  16. 如权利要求1所述的制备方法，其特征在于，所述步骤S3中真空度从2~4KPa增加到2~3Mpa，相对压强计，所述真空度的比值范围为1.2~200倍。
  17. 如权利要求1所述的制备方法，其特征在于，在每个真空度条件下，所述组织材料的处理时间为30秒~30分钟。
  18. 如权利要求1所述的制备方法，其特征在于，所述步骤S3中使用医疗级无尘布将浸泡处理后的所述组织材料的表面残留溶液吸除。
  19. 如权利要求1所述的制备方法，其特征在于，所述步骤S4中的密封包装过程如下：在相对湿度小于30%的环境或惰性环境将所述组织材料置入无液体的容器或包装袋进行包装。
  20. 如权利要求1所述的制备方法，其特征在于，所述的灭菌方式为EO灭菌、电子束辐射灭菌或伽马射线辐照灭菌。
  21. 一种干态动物源性胶原纤维组织材料，所述组织材料通过权利要求1-20中的任一制备方法制备得到。
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