WO2016180259A1 - 一种干态动物源性胶原纤维组织材料及其制备方法和生物假体 - Google Patents

一种干态动物源性胶原纤维组织材料及其制备方法和生物假体 Download PDF

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WO2016180259A1
WO2016180259A1 PCT/CN2016/080965 CN2016080965W WO2016180259A1 WO 2016180259 A1 WO2016180259 A1 WO 2016180259A1 CN 2016080965 W CN2016080965 W CN 2016080965W WO 2016180259 A1 WO2016180259 A1 WO 2016180259A1
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tissue material
preparation
solution
aqueous
tissue
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PCT/CN2016/080965
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English (en)
French (fr)
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董教明
陈叶萌
徐冠彪
程秀兰
陈国明
李�雨
罗七一
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上海微创心通医疗科技有限公司
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Priority to US15/571,953 priority Critical patent/US10383978B2/en
Priority to KR1020177033714A priority patent/KR102048010B1/ko
Priority to EP16792109.7A priority patent/EP3296391B1/en
Priority to JP2017557385A priority patent/JP6579588B2/ja
Priority to BR112017023732-6A priority patent/BR112017023732B1/pt
Publication of WO2016180259A1 publication Critical patent/WO2016180259A1/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
    • A61L27/3683—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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/3691—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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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

Definitions

  • the invention belongs to the field of medical biomaterials, and particularly relates to a dry animal-derived collagen fiber tissue material, a preparation method thereof and a biological prosthesis.
  • tissue materials are usually stored using chemical reagents such as glutaraldehyde and / or formaldehyde or The crosslinking treatment is carried out using the chemical reagent.
  • the tissue is typically stored in a dilute aqueous solution containing glutaraldehyde and/or formaldehyde to maintain the tissue components in a sterile environment and maintain a hydrated state.
  • glutaraldehyde remaining in the valve tissue after implantation promotes calcification of the valve.
  • glutaraldehyde treated and preserved biological tissue prostheses must undergo extensive cleaning to remove glutaraldehyde prior to implantation.
  • exposure of production workers, medical personnel, and patients to aldehyde storage solutions can cause significant harm to the body, requiring additional protective measures, resulting in increased costs and inconvenience.
  • medical personnel should make as little preparation as possible before surgery, and can easily obtain ready-to-use tissues and prostheses, which not only reduces the dyeing. The chance of bacteria or error also reduces implant time. Therefore, the development of a new dry storage method for biological tissues has broad development prospects and important application value.
  • the common biological tissue preservation method is cryopreservation and preservation, and the principle is to make the biological group
  • the water in the weave forms ice crystals at -80 ° C, and is lyophilized (vacuum dried) to obtain dry tissues.
  • freeze-dried biological tissues are inflexible due to the loss of moisture and hydrophilic solvents, and cannot prevent breakage, and the production cost is relatively high.
  • the formation of ice crystals also destroys the structure of the tissue, and the ability of the dry biological tissue to rehydrate is weak, and it usually takes several days to restore the original hydration state.
  • Chinese patent CN1306445A describes a method for treating tissue by first immersing biological tissue in a gradient-increasing polar organic solution (selected from methanol, ethanol, isopropanol, acetonitrile, acetone and methyl ethyl ketone), and then immersed in an aqueous glycerin solution. Or low molecular weight ( ⁇ 1000D) polyethylene glycol and 6000 ⁇ 15000D polyethylene glycol and heparin solution. Thereafter, the biological tissue was briefly immersed in an aqueous heparin solution for freezing and lyophilization.
  • a gradient-increasing polar organic solution selected from methanol, ethanol, isopropanol, acetonitrile, acetone and methyl ethyl ketone
  • Fumoto et al reported that the tissue was treated with a 57% aqueous glycerin solution and dried in an environment with a relative humidity of less than 28% for 6-8 hours. The size, shape and in vitro pulsation of the tissue after sterilization of ethylene oxide (EO) were performed. There is no significant change in flow performance. For details, see Fumoto H, Chen JF, Zhou Q, et al. Performance of bioprosthetic valves after glycerol dehydration, ethylene oxide sterilization, and rehydration. Innovations (Phila), 2011, 6(1): 32-36.
  • Chinese patent CN101965205A describes a method for dehydrating a dried biological tissue or prosthesis of 75% glycerol/25% ethanol.
  • Chinese patent CN103933612A 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, which treatment process maintains the tissue in a substantially dry state.
  • a polyol selected from one or a combination of: glycerol, propylene glycol, a derivative of glycerol and a derivative of propylene glycol
  • a C1-C3 alcohol selected from methanol
  • 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”. Residual water content in the tissue (including A water content greater than 30% will affect the EO sterilization effect resulting in incomplete sterilization).
  • the technical problem to be solved by the present invention is to provide a dry animal-derived collagen fiber tissue material, a preparation method thereof and a biological prosthesis, and the prepared dry animal-derived collagen fiber tissue material has good flexibility and no residual reagent.
  • the moisture content of the tissue material is controlled between 10% and 25% to facilitate subsequent sterilization.
  • the technical solution adopted by the present invention to solve the above technical problems is to provide a method for preparing a dry animal-derived collagen fiber tissue material, which comprises the following steps: (1) an animal source after cross-linking treatment by a crosslinking agent (1) immersing the washed tissue material in a non-aqueous alcohol solution for dehydration; (3) immersing the tissue material dehydrated by the non-aqueous alcohol solution in different concentrations The gradient saccharide aqueous solution is subjected to gradient dehydration; (4) the gradient dehydrated tissue material is taken out for drying; (5) the dried tissue material is sealed and packaged and then sterilized.
  • animal-derived collagen fiber tissue material in the step (1) is a heterologous or allogeneic biological tissue material.
  • the biological tissue material is a pericardium, a heart valve, a lamella, a pleura, a small intestinal submucosa, a dura mater, a dura mater, a ligament or a skin.
  • the crosslinking agent in the step (1) is one or more of glutaraldehyde, genipin, proanthocyanidins, and carbonized diimine.
  • the cleaning process in the step (1) is as follows: shaking with a salt solution of 5% to 30% (v/v) of isopropyl alcohol and/or ethanol at a temperature of 4 to 25 ° C for 3 to 60 minutes. Oscillation speed The rate is 50 to 150 rpm.
  • the salt solution is 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.
  • non-aqueous alcohol solution in the step (2) is a fatty alcohol solution having a molecular weight of less than 1000D.
  • non-aqueous alcohol solution is a polyether diol and/or a C 2 -C 6 fatty alcohol solution.
  • non-aqueous alcohol solution is polyethylene glycol, triethylene glycol, 1,2,6-hexanetriol, 1,2,4-butanetriol, 1,3-butanediol, 1, One or more of 4-butanediol, 1,5-pentanediol, glycerin, isopropanol, and ethanol.
  • the component in the non-aqueous alcohol solution comprises polyethylene glycol or glycerin, and the volume percentage of the polyethylene glycol or glycerin is 20% to 90%; or, in the non-aqueous alcohol solution
  • the component comprises polyethylene glycol and glycerin, and the percentage of the sum of the volume of the polyethylene glycol and glycerin is from 20% to 90%.
  • the polyethylene glycol has a number average molecular weight of 200 to 1,000.
  • the temperature of the non-aqueous alcohol solution in the step (2) is 20 to 37 ° C, and the immersion time of the tissue material in the dark-proof condition is 30 minutes to 24 hours.
  • the saccharide in the step (3) is a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide or a sugar alcohol sugar having a water absorption and a moisturizing type.
  • the saccharide is fructose, sucrose, trehalose, amorphous raffinose, chitosan and chitosan modified polysaccharide, sorbitol or mannitol.
  • the gradient dehydration process in the step (3) is as follows: the tissue material dehydrated by the non-aqueous alcohol solution is sequentially immersed in different concentrations of the aqueous solution of the saccharide, and the temperature of the aqueous solution of the saccharide is 4 to 37 ° C. In the dark, the immersion time is 5 minutes to 48 hours.
  • the aqueous solution of the saccharide with different concentration gradients in the step (3) is an aqueous solution of sucrose having a concentration of 30%, 40%, 50%, 55%, 60%, 65% (w/v), respectively; 50%, 60%, 70%, 75%, 80%, 85% (w/v) fructose, trehalose, amorphous cotton
  • aqueous solution of sugar, chitosan, chitosan modified polysaccharide, sorbitol or mannitol is an aqueous solution of sucrose having a concentration of 30%, 40%, 50%, 55%, 60%, 65% (w/v), respectively; 50%, 60%, 70%, 75%, 80%, 85% (w/v) fructose, trehalose, amorphous cotton
  • the tissue material after the gradient dehydration is taken out and then placed in a fiber desiccant for drying; or the temperature is controlled from 20 ° C to 37 ° C in a dark environment, and the air is allowed to stand for 10 minutes to 24 hours.
  • the fiber drying agent is a fiber drying agent original sheet, a folded fiber drying agent, a bag fiber drying agent, a columnar fiber drying agent or a coated fiber drying agent.
  • the sealed packaging process is as follows: the dried tissue material is placed in a packaging container for sealing packaging in an environment with a relative humidity of less than 30% or an inert gas atmosphere.
  • the sterilization is performed by 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 obtained by the above preparation method.
  • a third technical solution adopted by the present invention to solve the above technical problems is to provide a bioprosthesis prepared from the above-mentioned dry animal-derived collagen fiber tissue material.
  • the present invention has the following beneficial effects: the dry animal-derived collagen fiber tissue material provided by the invention, the preparation method thereof and the biological prosthesis have the following effects compared with the prior art: 1) preparation The method is simple, the source of raw materials is wide and low, and the large-scale production can reduce the medical cost; 2) the biological tissue after washing with isopropyl alcohol or ethanol can preliminarily remove the free cross-linking agent in the tissue (such as glutaraldehyde and Formaldehyde) solution to reduce toxicity caused by residual agent such as aldehydes; 3) Biological and physical joint drying method using non-aqueous alcohol solution dehydration, saccharide hypertonic water absorption and fiber desiccant water absorption The material has good flexibility, is not easy to curl, and the moisture content of the tissue can be effectively controlled.
  • the residual polyol chemical in the biological tissue is removed to make the tissue more biocompatible; 4) the sugar (especially sucrose) is high.
  • Infiltration can not only remove water from biological tissues, but also further eliminate residual cross-linking agents (such as formaldehyde and glutaraldehyde) in biological tissues, and improve the biological group.
  • Bio-safety and reduced potential for tissue calcification, and because these low molecular weight sugar gradient and can penetrate tissue dewatering methods used to prevent moisture lost due to rapid tissue Loss of voids and fractures has a strong protective effect on the composition and structure of the tissue; 5)
  • the light-shielding conditions are favorable for the biological tissue to maintain the original composition and structure, and prevent dry tissue.
  • the prepared biological tissue material and the prosthesis thereof have good biocompatibility, flexible material, good mechanical properties, safe and reliable use, easy clinical operation, and rehydration in physiological saline.
  • the speed is fast (generally, the original hydration state can be restored in about 5 minutes).
  • FIG. 1 is a flow chart showing the preparation of a dry animal-derived collagen fiber tissue material according to an embodiment of the present invention
  • Figure 2 is an electron micrograph (1200x) of bovine pericardium tissue after dry treatment.
  • Fig. 3 is a photograph showing histological (HE) staining results of a control bovine pericardium tissue piece which was not subjected to dry treatment in Example 1 of the present invention, which was taken 4 weeks after subcutaneous implantation in Wistar rats.
  • HE histological
  • Fig. 4 is a photograph showing histological (HE) staining results of the dried bovine pericardial tissue sheet prepared in Example 1 of the present invention at 4 weeks after subcutaneous implantation in Wistar rats.
  • HE histological
  • a method for preparing a dry animal-derived collagen fiber tissue material comprises the following steps:
  • the animal-derived collagen fiber tissue material cross-linked by the cross-linking agent is a heterogeneous or allogeneic biological tissue material cross-linked by a crosslinking agent, such as a pericardium, a heart valve, a laminar, Pleural, submucosal, dura mater, dura mater, ligament and skin.
  • a crosslinking agent such as a pericardium, a heart valve, a laminar, Pleural, submucosal, dura mater, dura mater, ligament and skin.
  • the cross-linking agent used in the treatment of the animal-derived collagen fiber tissue material in the present invention is not particularly limited, and examples thereof include common glutaraldehyde, and new cross-linking agents such as Genipin, proanthocyanidins, and carbonized diimine. , or a mixture of the above crosslinkers.
  • the method for crosslinking the cross-linking agent to treat the animal-derived collagen fiber tissue material of the present invention is not particularly limited, and is, for example, treated by a chemical immersion method and treated by a steam method.
  • the concentration of the crosslinking agent in the treatment of the animal-derived collagen fibrous tissue material of the present invention is not particularly limited.
  • the washing means washing with a salt solution of 5% to 30% (v/v) of isopropyl alcohol and/or ethanol at 4 to 25 ° C for 3 to 60 minutes at an oscillation rate of 50 to 150 rpm.
  • the 5% to 30% (v/v) salt solution of isopropanol and/or ethanol as used herein means that the salt solution is a 5% to 30% (v/v) solution of isopropoxide, or It is a 5% to 30% (v/v) ethoxide solution, or a 5% to 30% (v/v) solution of isopropanol and ethanolate, and the ratio of isopropanol to ethanol is arbitrary.
  • the salt solution is physiological saline, or 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 (Hanks solution without calcium and magnesium ions, GIBCO).
  • the non-aqueous alcohol solution is a low molecular weight fatty alcohol ( ⁇ 1000 D), preferably a polyether diol (structure is OH-(RO-) n -R-OH, R is an alkylene group, Preferred is a C2-C6 alkylene group) and/or a C 2 -C 6 fatty alcohol (for example, a C 2 -C 6 monohydric alcohol, a C 2 -C 6 diol, a C 2 -C 6 triol, C 2 ⁇ C 6 tetrahydric alcohol), more preferably polyethylene glycol, triethylene glycol, 1,2,6-hexanetriol, 1,2,4-butanetriol, 1,3-butanediol, 1 One or several of 4-butanediol, 1,5-pentanediol, glycerin, isopropanol, ethanol, and the like.
  • a polyether diol structure is OH-(RO-) n -R-OH
  • the polyethylene glycol has a number average molecular weight of 200 to 1,000.
  • the polyethylene glycol is one of polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800, polyethylene glycol 1000 or A variety.
  • the amount of each component added is not particularly limited.
  • the mixture has two components, each component having a volume percentage of 5 to 95%; the mixture has three components, each component having a volume percentage of 5 to 90%; and the mixture having four The components, the volume percentage of each component is 5-85%; there are five components in the mixture, and the volume percentage of each component is 5 to 80%.
  • the components in the mixed solution include polyethylene glycol or glycerin, and the volume percentage is 20% ⁇ 90%;
  • the components in the mixed solution include polyethylene glycol and glycerin, and the combined volume of polyethylene glycol and glycerin is 20% to 90% by volume of the mixed liquid, and the ratio of polyethylene glycol to glycerin is arbitrary.
  • the immersion conditions in which the tissue material is immersed in the non-aqueous alcohol solution are an immersion temperature of 20 to 37 ° C, and an immersion time of 30 minutes to 24 hours, preferably 1 to 12 hours, in the dark.
  • step S3 The tissue material treated in step S2 is sequentially immersed in a different concentration gradient of the aqueous sugar solution.
  • the saccharide is a low molecular weight sugar having a water absorption and a moisturizing type such as a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide, and a sugar alcohol, for example, fructose, sucrose, trehalose, raffinose (non- Crystalline), chitosan, chitosan modified polysaccharide, sorbitol or mannitol.
  • a moisturizing type such as a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide, and a sugar alcohol, for example, fructose, sucrose, trehalose, raffinose (non- Crystalline), chitosan, chitosan modified polysaccharide, sorbitol or mannitol.
  • the immersing of the tissue material in the saccharide aqueous solution of different concentration gradients means that the tissue material is subjected to gradient dehydration, and the tissue material is sequentially immersed in different concentrations of the saccharide aqueous solution, and the immersion temperature is 4 to 37 ° C.
  • the immersion time is from 5 minutes to 48 hours, preferably from 30 minutes to 24 hours, more preferably from 1 to 12 hours, in the dark.
  • the different concentration gradients of the aqueous sugar solution are aqueous solutions of sucrose having a concentration of 30%, 40%, 50%, 55%, 60%, and 65% (w/v), respectively;
  • the aqueous solution of the different concentration gradients is 50%, 60%, 70%, 75%, 80%, 85% (w/v) of fructose, trehalose, amorphous cotton.
  • the fiber drying agent is not particularly limited and may be selected from the group consisting of a fiber desiccant original sheet, a folded fiber desiccant, a baglet fiber desiccant, a columnar fiber desiccant, and a coated fiber desiccant, preferably a coated fiber.
  • a fiber desiccant original sheet a folded fiber desiccant
  • a baglet fiber desiccant a baglet fiber desiccant
  • a columnar fiber desiccant preferably a coated fiber.
  • a coated fiber desiccant preferably a coated fiber.
  • the dry clean environment refers to a 10,000-class or a hundred-level purification workshop environment having a humidity of less than 20%.
  • the drying condition in the clean environment is 20 ° C to 37 ° C in a dark environment, and the drying time is 10 minutes to 24 hours, preferably 1 hour to 8 hours.
  • the manner in which the clean environment is obtained is not particularly limited, and the moisture content in the air can be lowered by the desiccant, and the air can be ventilated by dry air.
  • the sealed package refers to packaging the above-mentioned tissue material and/or its biological prosthesis into a container such as a liquid-free packaging bag under an environment having an relative humidity of less than 30% or an inert environment such as nitrogen, argon or the like. Sealed packaging.
  • the sterilization refers to sterilization by EO sterilization (Ethylene Oxide), electron beam radiation sterilization or gamma ray irradiation.
  • EO sterilization Ethylene Oxide
  • electron beam radiation sterilization or gamma ray irradiation.
  • Subcutaneous implantation test and pathological observation in rats male young Wistar rats were used. After anesthesia, an incision was made along the back of the rat under aseptic conditions. The tissue piece with a size of 15 mm ⁇ 15 mm was inserted into the incision and the incision was made. day. After 28 days, the cells were sacrificed by carbon dioxide gas suffocation, the tissue pieces were taken out, fixed with 10% neutral formalin, embedded in paraffin, cut into 0.4 micron thick slices, dewaxed by xylene, dehydrated by series of alcohol, hematoxylin-eosin Dyeing, observing fiber and inflammation.
  • tissue material was fixed with 2.5% glutaraldehyde solution, ethanol was dehydrated step by step, dried, vacuum sprayed with gold, and the surface morphology and fiber arrangement of the scanning electron microscope were observed.
  • tissue material is cut into a sheet shape, and it is pulled off on a universal material testing machine. The maximum tensile force measured is divided by the cross-sectional area of the test material, which is the tensile strength of the tissue material. Indicates the tensile strength of the material.
  • Rehydration time of biological tissue material The treated 15 mm ⁇ 15 mm tissue samples were immersed in physiological saline at 37 ° C, and the tissue materials at different time points were taken out, and calculated according to "2. Determination of water content of biological tissue materials" Water content, when the water content of the tissue reaches 70% or more, record the corresponding soaking time. between.
  • Thermal shrinkage temperature of biological tissue material Six 50 mm ⁇ 3 mm test strips were cut on the tissue sheet, and the heat shrinkage temperature was measured on a leather shrinkage temperature measuring instrument using distilled water as a medium.
  • bovine pericardium tissue was obtained at the local slaughterhouse, and after being stripped, trimmed and washed, it was fixed with 0.625% (v/v) glutaraldehyde (Sigma-Aldrich Co. LLC) solution for more than 3 days to obtain cross-linking with glutaraldehyde. Treated cattle pericardium.
  • the bovine pericardium was then immersed in 70% (v/v) polyethylene glycol 200 (Shanghai Jingchun Biochemical Technology Co., Ltd.) and 30% (v/v) 1,3-butanediol (Sigma-Aldrich Co In a blue cap bottle of a mixed alcohol solution of .LLC.), the blue cap bottle was completely wrapped with tin foil paper and immersed for 4 hours at 20 °C. Then, the bovine pericardium was immersed at 20 °C into sucrose with a concentration of 30%, 40%, 50%, 55%, 60%, 65% (w/v) completely wrapped with tin foil.
  • the immersion time is 30 minutes each time.
  • the bovine pericardium is taken out and placed on a coated fiber desiccant (Shanghai Hengyuan Polymer Material Co., Ltd.) for drying.
  • a coated fiber desiccant Shanghai Hengyuan Polymer Material Co., Ltd.
  • place the dry bovine pericardium into the dialysis bag DuPont China Group Co., Ltd.), sealed, EO sterilization.
  • Fig. 2 is an electron micrograph of the prepared bovine pericardium tissue after dry treatment. It can be seen from Fig. 2 that after treatment by the dry method of the present invention, the collagen fibers in the bovine pericardium are arranged neatly, without obvious breakage, and arranged in a wave shape, and the structure is compact and continuous.
  • the prepared dry bovine pericardium tissue has a water content of 17.3 ⁇ 1.8%, a rehydration time of 5.37 ⁇ 0.42 minutes, and a heat shrinkage temperature of 86.67 ⁇ 1.35. °C, the cytotoxicity is grade 1, and the maximum tensile strength is 12.5 ⁇ 6.6N.
  • the untreated bovine pericardium and the dried and sterilized bovine pericardium were implanted into Wistar rats for 4 weeks.
  • the results showed that the control bovine pericardial tissue was harvested 4 weeks after subcutaneous implantation in Wistar rats.
  • the results of the (HE) staining showed that the fiber direction was relatively consistent, the continuity was good, and it was wavy.
  • scattered inflammatory cells were found in the pericardial tissue, and new capillary capillaries and a large number of inflammatory cells were observed in the unilateral loose connective tissue (see Figure 3). .
  • the histological (HE) staining of the dried bovine pericardial tissue in 4 weeks after subcutaneous implantation in Wistar rats showed that the fiber in the bovine pericardium was relatively consistent, with good continuity, wavy, pericardium. There were no visible inflammatory cells and inflammatory cells infiltrating in the tissues, and no calcifications were found in the pericardial tissues (see Figure 4). It can be seen that the bovine pericardium tissue prepared by the invention has good biocompatibility and can maintain the original structure and state of the tissue.
  • the porcine aortic valve was obtained at a local slaughterhouse. After washing, it was fixed with 0.625% (v/v) glutaraldehyde solution for more than 3 days to obtain a porcine aortic valve after glutaraldehyde cross-linking treatment.
  • the treated porcine aortic valve was washed with a physiological saline solution of 20% (v/v) ethanol (National Pharmaceutical Group Chemical Co., Ltd.) at 20 ° C with shaking at 100 rpm, and washed 3 to 5 times for 3 to 5 minutes each time.
  • the porcine aortic valve was then immersed in a blue cap bottle containing 100% glycerol (Scientia Pharmaceutical Group Chemical Co., Ltd.) solution, and the blue cap bottle was completely wrapped with tin foil paper and immersed for 3 hours at 20 °C. After the time has elapsed, the porcine aortic valve was sequentially immersed at 4 ° C into fructose at 50%, 60%, 70%, 75%, 80%, 85% (w/v) completely wrapped with tin foil. In the blue cap bottle of the aqueous solution, the immersion time was 30 minutes each time. Thereafter, in a dry clean environment, the porcine aortic valve is removed and placed on a coated fiber desiccant for drying.
  • glycerol Stemethanol
  • 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 15.7 ⁇ 2.1%, a rehydration time of 4.85 ⁇ 0.78 minutes, a heat shrinkage temperature of 85.59 ⁇ 2.46°C, a cytotoxicity of grade 1, and a maximum tensile strength of 11.9 ⁇ 4.3N.
  • the submucosal tissue of the small intestine of the pig was obtained at the local slaughterhouse, and after being stripped, trimmed and washed, it was fixed with 0.625% (v/v) glutaraldehyde solution for more than 3 days to obtain the submucosal tissue of the small intestine of the pig which was cross-linked by glutaraldehyde.
  • the treated porcine small intestine submucosal tissue was cut into small pieces (30 mm ⁇ 50 mm), and the submucosal tissue of the small intestine of the small intestine was washed with a physiological saline solution of 10% (v/v) isopropanol at 25 ° C, and washed 3 to 5 Once, wash for 3 to 5 minutes each time.
  • the porcine small intestine submucosal tissue was then immersed in 50% (v/v) 1,2,6-hexanetriol (Shanghai Jingchun Biochemical Technology Co., Ltd.), 30% (v/v) ethanol and 20% ( v/v)
  • 50% (v/v) 1,2,6-hexanetriol Shanghai Jingchun Biochemical Technology Co., Ltd.
  • 30% (v/v) ethanol 30%
  • 20% ( v/v) In the blue cap bottle of the mixed alcohol solution of polyethylene glycol 400 (Shanghai Jingchun Biochemical Technology Co., Ltd.), the blue cap bottle was completely wrapped with tin foil paper, and immersed at 20 ° C for 2 hours.
  • the submucosal tissue of the small intestine of the pig is immersed at 20 ° C in turn at a concentration of 30%, 40%, 50%, 55%, 60%, 65% (w/v) completely wrapped with tin foil.
  • the immersion time was 20 minutes each time.
  • the submucosal tissue of the small intestine of the pig is taken out and placed on a coated fiber desiccant for drying.
  • 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 16.5 ⁇ 1.6%, a rehydration time of 4.73 ⁇ 0.91 minutes, a heat shrinkage temperature of 87.34 ⁇ 1.15°C, a cytotoxicity of grade 1, and a maximum tensile strength of 12.7 ⁇ 5.2N.
  • the pig pericardium tissue was obtained at a local slaughterhouse and fixed with 0.625% (v/v) glutaraldehyde solution for more than 3 days after fat stripping, trimming and washing. Cut the fixed pig pericardium into small pieces (30mm ⁇ 50mm), wash the pig pericardium with 15% (v / v) isopropanol physiological saline at 20 ° C shaking at 100 rpm, wash 3 to 5 times, each wash 3 ⁇ 5 minutes. The pig pericardium was then immersed in a blue cap bottle containing a mixed alcohol solution of 75% glycerol and 25% isopropanol, and the blue cap bottle was completely wrapped with tin foil paper and immersed for 3 hours at 20 °C.
  • the pig pericardium is immersed at 20 ° C in the blue of the sucrose aqueous solution with a concentration of 30%, 40%, 50%, 55%, 60%, 65% (w/v) completely wrapped with tin foil.
  • the immersion time is 10 minutes each time.
  • the pig's pericardium is removed and placed on a coated fiber desiccant for drying.
  • the dry pig pericardium is placed in a bottle, sealed, and sterilized by electron beam irradiation.
  • the prepared dry pig pericardium tissue has a water content of 18.0 ⁇ 1.2%, a rehydration time of 5.15 ⁇ 0.36 minutes, a heat shrinkage temperature of 87.84 ⁇ 1.66°C, a cytotoxicity of 1 grade, and a maximum tensile strength of 14.2 ⁇ . 5.8N.

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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和生物假体，所述制备方法包括如下步骤：(1)对经过交联剂交联处理后的动物源性胶原纤维组织材料进行清洗；(2)将清洗后的所述组织材料浸没在非水性醇溶液中进行脱水；(3)将经过非水性醇溶液脱水处理的所述组织材料依次浸没在不同浓度梯度的糖类水溶液中进行梯度脱水；(4)取出所述组织材料进行干燥；(5)将干燥后的所述组织材料密封包装后进行灭菌。制备方法简单、原料来源广泛且低廉，可降低医疗成本；并降低由于醛类等交联剂残留引起的毒性；得到的生物组织材料柔韧性好，不易卷曲，组织含水量可得到有效控制，同时去除生物组织中残留的多元醇化学试剂使组织具有更优良的生物相容性。

Description

一种干态动物源性胶原纤维组织材料及其制备方法和生物假体 技术领域
本发明属于医学生物材料领域，具体涉及一种干态动物源性胶原纤维组织材料及其制备方法和生物假体。
背景技术
目前，临床上常用的生物瓣膜和经导管主动脉瓣膜，以及生物补片，如牛心包，小肠粘膜下层等所用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。组织一般保存在包含戊二醛和/或甲醛的稀释水溶液中，使组织成分保持无菌环境和保持水合状态。然而，大量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。在贮存过程中消除醛类试剂可有效降低瓣膜组织的钙化。Mirzaie等研究发现采用人血清保存溶液代替戊二醛溶液贮存猪主动脉瓣膜，瓣膜钙含量可降低50％左右；此外，戊二醛具有较高毒性，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；具体请参见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，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。同样，生产工人、医疗人员和患者暴露在醛类保存溶液中对身体产生显著的危害，需要额外的防护措施，从而导致费用的增加和不便。但是需要考虑的是，在植入的生物组织材料和其组成的假体时，医疗人员应当尽可能在手术前进行最少的准备，能够容易得到即用的组织和假体，这不仅降低了染菌或误差机率，也减少了植入时间。因此，开发一种新的生物组织干态贮存方式具有广阔的发展前景和重要的应用价值。
目前常见的生物组织保存方法是低温冷冻干燥保存，其原理是使生物组 织中的水分在-80℃形成冰晶，再低压冻干(真空干燥)得到干态组织。然而，这种冻干的生物组织由于失去水分和亲水性的溶剂是不柔韧的，并且不能防止断裂，生产成本也相对较高。同时，冰晶的形成也会破坏组织的结构，且这种干态的生物组织再水化的能力较弱，通常需要几天才能恢复原先的水合状态。
中国专利CN1306445A描述了一种溶液处理组织的方法，首先将生物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)，然后被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。此后，生物组织被简短浸没在肝素水溶液中冷冻和冻干。然而，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，另外，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。
Fumoto等报道了采用57％甘油水溶液处理组织并在相对湿度小于28％的环境中干燥6～8小时，环氧乙烷(Ethylene oxide,EO)灭菌后组织的尺寸大小、形状和瓣膜体外脉动流性能没有明显变化。具体请参见Fumoto H,Chen JF,Zhou Q,et al.Performance of bioprosthetic valves after glycerol dehydration,ethylene oxide sterilization,and rehydration.Innovations(Phila),2011,6(1):32-36.
中国专利CN101965205A描述了一种75％甘油/25％乙醇脱水干燥的生物组织或假体的方法。
中国专利CN103933612A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，这种处理方法能够使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试剂残留毒性、组织形态变化和费用高等缺点，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变化等方面仍需要进一步改进。
发明内容
本发明所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和生物假体，制备的干态动物源性胶原纤维组织材料具有较好的柔顺性，无残留试剂，组织材料含水量控制在10％～25％之间，以利于后续灭菌。
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，其中，包括如下步骤：(1)对经过交联剂交联处理后的动物源性胶原纤维组织材料进行清洗；(2)将清洗后的所述组织材料浸没在非水性醇溶液中进行脱水；(3)将经过非水性醇溶液脱水处理的所述组织材料依次浸没在不同浓度梯度的糖类水溶液中进行梯度脱水；(4)取出梯度脱水后的所述组织材料进行干燥；(5)将干燥后的所述组织材料密封包装后进行灭菌。
进一步地，所述步骤(1)中动物源性胶原纤维组织材料为异种异体或同种异体的生物组织材料。
进一步地，所述生物组织材料为心包、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(1)中的交联剂为戊二醛、京尼平、原花青素、炭化二亚胺中的一种或多种。
进一步地，所述步骤(1)中的清洗过程如下：用体积百分比5％～30％(v/v)的异丙醇和/或乙醇的盐溶液在4～25℃下振荡清洗3～60分钟，振荡速 率为50～150rpm。
进一步地，所述盐溶液为生理盐水、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。
进一步地，所述步骤(2)中的非水性醇溶液为分子量小于1000D的脂肪醇溶液。
进一步地，所述非水性醇溶液为聚醚二醇和/或C2～C6脂肪醇溶液。
进一步地，所述非水性醇溶液为聚乙二醇、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、甘油、异丙醇和乙醇中的一种或多种。
进一步地，所述非水性醇溶液中的组分包含聚乙二醇或甘油，且所述聚乙二醇或甘油的体积百分比为20％～90％；或者，所述非水性醇溶液中的组分包含聚乙二醇和甘油，且所述聚乙二醇与甘油的体积之和的百分比为20％～90％。
进一步地，所述聚乙二醇的数均分子量为200～1000。
进一步地，所述步骤(2)中非水性醇溶液的温度为20～37℃，所述组织材料在避光条件下浸没时间为30分钟～24小时。
进一步地，所述步骤(3)中的糖类为具有吸水和保湿型的单糖、二糖、三糖、多糖或糖醇类糖。
进一步地，所述糖类为果糖、蔗糖、海藻糖、非晶态的棉子糖、壳聚糖及壳聚糖改性的多糖、山梨糖醇或甘露糖醇。
进一步地，所述步骤(3)中的梯度脱水过程如下：依次将经过非水性醇溶液脱水处理的所述组织材料浸入到不同浓度的糖类水溶液，所述糖类水溶液温度为4～37℃，避光条件下每次浸没时间为5分钟～48小时。
进一步地，所述步骤(3)中不同浓度梯度的糖类水溶液为浓度分别为30％，40％，50％，55％，60％，65％(w/v)的蔗糖水溶液；或者浓度分别为50％，60％，70％，75％，80％，85％(w/v)的果糖、海藻糖、非晶态的棉子 糖、壳聚糖、壳聚糖改性的多糖、山梨糖醇或甘露糖醇的水溶液。
进一步地，所述步骤(4)中取出梯度脱水后的所述组织材料后放入纤维干燥剂进行干燥；或者在避光环境中控制温度20℃～37℃，晾放10分钟～24小时。
进一步地，所述的纤维干燥剂为纤维干燥剂原片、折叠纤维干燥剂、袋片纤维干燥剂、柱状纤维干燥剂或覆膜纤维干燥剂。
进一步地，所述的密封包装过程如下：在相对湿度小于30％的环境或惰性气体环境下将干燥后的所述组织材料放入到包装容器中进行密封包装。
进一步地，所述的灭菌采用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，由上述制备方法制取。
本发明为解决上述技术问题而采用的第三种技术方案是提供一种生物假体，由上述干态动物源性胶原纤维组织材料制取。
本发明对比现有技术有如下的有益效果：本发明提供的一种干态动物源性胶原纤维组织材料及其制备方法和生物假体，与现有技术相比，具有以下效果：1)制备方法简单、原料来源广泛且低廉，规模化生产可降低医疗成本；2)采用的异丙醇或乙醇的盐水清洗固定后的生物组织可初步去除组织中游离的交联剂(例如戊二醛和甲醛)溶液，降低由于醛类等交联剂残留引起的毒性；3)采用了非水性醇溶液脱水、糖类高渗性吸水和纤维干燥剂吸水的化学和物理联合干燥方法，得到的生物组织材料柔韧性好，不易卷曲，组织含水量可得到有效控制，同时去除生物组织中残留的多元醇化学试剂使组织具有更优良的生物相容性；4)采用的糖类(特别是蔗糖)高渗吸水不仅能脱除生物组织中的水分，还能进一步消除生物组织内残留的交联剂(例如甲醛和戊二醛)，提高生物组织的生物安全性和降低组织钙化的潜能，且由于这些低分子量的糖能渗透组织中和采用的梯度脱水方式，防止组织由于水分的快速丢 失而形成空洞和断裂，对组织的组成成分和结构具有较强的保护作用；5)在干态处理过程中，采用的避光条件有利于生物组织保持原先的组成成分和结构，防止干组织的氧化和结构的破坏；6)所制备的生物组织材料及其构成的假体的生物相容性好、材料柔韧、力学性能好，使用安全可靠，易于临床手术操作，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)等优点。
附图说明
图1为本发明实施例中干态动物源性胶原纤维组织材料的制备流程图；
图2为干态处理后的牛心包组织的电子显微镜照片(1200x)。
图3为采用本发明实施例1中未经干态处理的对照牛心包组织片在Wistar大鼠皮下植入后4周取材的组织学(HE)染色结果照片。
图4为采用本发明实施例1中所制备的干态处理的牛心包组织片在Wistar大鼠皮下植入后4周取材的组织学(HE)染色结果照片。
具体实施方式
下面结合附图和实施例对本发明作进一步的描述。
请参见图1，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，包括以下步骤：
S1:对经过交联剂交联处理后的动物源性胶原纤维组织材料进行清洗。
根据本发明，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，炭化二亚胺等新型交联剂，或者以上交联剂的混合。
本发明对交联剂交联处理动物源性胶原纤维组织材料的方法没有特别的限定，例如采用化学浸没方法处理，采用蒸汽方法处理。
本发明对处理动物源性胶原纤维组织材料时交联剂的浓度没有特别的限制。
所述的清洗是指用5％～30％(v/v)的异丙醇和/或乙醇的盐溶液在4～25℃下振荡清洗3～60分钟，振荡速率为50～150rpm。这里所述的5％～30％(v/v)的异丙醇和/或乙醇的盐溶液是指，所述盐溶液为5％～30％(v/v)的异丙醇盐溶液，或者为5％～30％(v/v)的乙醇盐溶液，或者为5％～30％(v/v)的异丙醇和乙醇盐溶液，且异丙醇和乙醇比例任意。
所述的盐溶液为生理盐水、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(无钙镁离子的Hanks液，GIBCO公司)。
S2:将清洗后的组织材料浸没在非水性醇溶液(非含水)中进行脱水；
根据本发明，所述的非水性醇溶液是低分子量的脂肪醇(＜1000D)，优选为聚醚二元醇(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脂肪醇(例如，C2～C6一元醇，C2～C6二元醇，C2～C6三元醇，C2～C6四元醇)，更优选为聚乙二醇、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、甘油、异丙醇、乙醇等中的一种或数种。
所述的聚乙二醇的数均分子量为200～1000。例如，所述的聚乙二醇为聚乙二醇200，聚乙二醇300、聚乙二醇400、聚乙二醇600、聚乙二醇800、聚乙二醇1000中的一种或多种。
所述的非水性醇溶液如果是混合液，则各组分的添加量没有特别的限制。优选，混合液中有两种组分，每种组分的体积百分比为5～95％；混合液中有三种组分，每种组分的体积百分比为5～90％；混合液中有四种组分，每种组分的体积百分比为5～85％；混合液中有五种组分，每种组分的体积百分比为5～80％。优选，混合液中的组分包括聚乙二醇或甘油，且体积百分比为20％～ 90％；优选，混合液中的组分包括聚乙二醇和甘油，且聚乙二醇和甘油的体积之和占混合液的体积百分比为20％～90％，聚乙二醇和甘油的比例任意。
根据本发明，所述的组织材料浸没在非水性醇溶液中的浸没条件是浸没温度为20～37℃，避光条件下浸没时间为30分钟～24小时，优选1～12小时。
S3:将步骤S2处理的组织材料依次浸没在不同浓度梯度的糖类水溶液中。
根据本发明，所述的糖类为单糖、二糖、三糖、多糖和糖醇类等低分子量的具有吸水和保湿型的糖，例如，果糖、蔗糖、海藻糖、棉子糖(非晶态)、壳聚糖、壳聚糖改性的多糖、山梨糖醇或甘露糖醇。
根据本发明，所述的组织材料依次浸没在不同浓度梯度的糖类水溶液中是指对组织材料进行梯度脱水，依次将组织材料浸入到不同浓度的糖类水溶液，浸没温度为4～37℃，避光条件下每次浸没时间为5分钟～48小时，优选30分钟～24小时，更优选地1～12小时。在一些实施例中，所述的不同浓度梯度的糖类水溶液为浓度分别为30％，40％，50％，55％，60％，65％(w/v)的蔗糖水溶液；在另外一些实施例中，所述的不同浓度梯度的糖类水溶液为浓度分别为50％，60％，70％，75％，80％，85％(w/v)的果糖、海藻糖、非晶态的棉子糖、壳聚糖、壳聚糖改性的多糖、山梨糖醇或甘露糖醇的水溶液。
S4:取出组织材料放入含有纤维干燥剂和/或干燥的洁净环境中干燥。
根据本发明，所述的纤维干燥剂没有特别的限制，可以选自纤维干燥剂原片、折叠纤维干燥剂、袋片纤维干燥剂、柱状纤维干燥剂和覆膜纤维干燥剂，优选覆膜纤维干燥剂。
根据本发明，所述的干燥的洁净环境是指湿度小于20％的万级或百级净化车间环境。所述的洁净环境中干燥的条件是避光环境下温度为20℃～37℃，干燥时间为10分钟～24小时，优选地1小时～8小时。本发明中对洁净环境的获得方式没有特别的限制，可以通过干燥剂降低空气中的水分含量，可以是通过干燥空气换气。
S5:密封包装后进行灭菌。
根据本发明，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、氩气等下将上述组织材料和/或其生物假体包装到无液体的包装袋等容器中进行密封包装。
根据本发明，所述的灭菌是指采用EO灭菌(Ethylene Oxide；环氧乙烷)、电子束辐射灭菌或伽马射线辐照灭菌。
测试方法
本发明方法制备得到的干态动物源性胶原纤维组织材料的评价标准及其方法：
1.大鼠皮下植入试验和病理观察：选用雄性幼年Wistar大鼠，麻醉后在无菌条件下沿大鼠背部做出切口，将大小为15mm×15mm组织片植入后缝合切口，饲养28天。28天后，用二氧化碳气体窒息处死，取出组织片，用10％中性福尔马林固定，石蜡包埋，切成0.4微米厚的薄片，经二甲苯脱蜡、系列酒精脱水，苏木素一伊红染色，观察纤维和炎症状况。
2.生物组织材料含水量的测定：将湿态样品夹在两层干滤纸中间，滤纸上压一重50g的物体30s，测得样品湿重；将样品真空冷冻干燥24h测得干重。按含水量＝(湿重-干重)/干重×100％计算样品的含水量。
3.生物组织材料表面形貌：组织材料用2.5％戊二醛溶液固定，乙醇逐级脱水，干燥，真空喷金，扫描电镜组织表面形貌和纤维排列情况。
4.生物组织材料拉伸强度的测定：将组织材料裁成板材状，在万能材料试验机上将其拉断，所测的最大拉力除以试材的截面积，为组织材料的拉伸强度，表示材料的抗拉能力。
5.生物组织材料的再水化时间：将处理后的15mm×15mm的组织样片浸入37℃生理盐水中，将不同时间点的组织材料取出，按“2.生物组织材料含水量的测定”计算含水量，当组织含水量达到70％以上时，记下对应的浸泡时 间。
6.生物组织材料的热皱缩温度：组织片上裁取6根50mm×3mm的试条,以蒸馏水为介质,在皮革收缩温度测定仪上测定其热皱缩温度。
7.生物组织材料体外细胞毒性实验：根据GB16886.5-2003和GB/T 14233.2-2005_8的方法，采用浸提液和MTT的方法，对材料的细胞毒性进行测试。
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0.625％(v/v)戊二醛(Sigma-Aldrich Co.LLC.)溶液固定3天以上，得到经过戊二醛交联处理后的牛心包。将处理后的牛心包裁剪成小片(15mm×15mm)，用10％(v/v)异丙醇(国药集团化学试剂有限公司)的生理盐水(山东康宁药业有限公司，批号：A14100807)在20℃下100rpm振荡清洗牛心包组织小片，清洗3～5次，每次清洗3～5分钟。之后将牛心包浸没在含70％(v/v)的聚乙二醇200(上海晶纯生化科技股份有限公司)和30％(v/v)1,3-丁二醇(Sigma-Aldrich Co.LLC.)的混合醇溶液的蓝盖瓶中，用锡箔纸完全包裹蓝盖瓶，20℃下浸没4小时。然后，将牛心包在20℃下依次浸入到用锡箔纸完全包裹的、浓度分别为30％，40％，50％，55％，60％，65％(w/v)的蔗糖(上海晶纯生化科技股份有限公司)水溶液的蓝盖瓶中，每次浸没时间为30分钟。之后，在干燥的洁净环境中，再将牛心包取出，放到覆膜纤维干燥剂(上海衡元高分子材料有限公司)上进行干燥。最后，在干燥的洁净环境中，将干态的牛心包放入透析袋(
Figure PCTCN2016080965-appb-000001
杜邦中国集团有限公司)中，密封，EO灭菌。
图2为所制备的干态处理后的牛心包组织的电子显微镜照片。从图2中可以看出，经本发明的干态方法处理后，牛心包组织中的胶原纤维排列整齐，无明显断裂，呈波浪状排列，结构紧凑连续。所制备的干态牛心包组织的含水量为17.3±1.8％，再水化时间为5.37±0.42分钟，热皱缩温度为86.67±1.35 ℃，细胞毒性为1级，最大拉伸强度为12.5±6.6N。将未经处理的牛心包和经过干态处理后灭菌的牛心包皮下植入Wistar大鼠4周时间，结果发现：对照牛心包组织片在Wistar大鼠皮下植入后4周取材的组织学(HE)染色结果照片，纤维走向相对一致，连续性好，呈波浪状，但心包组织中可见散在炎细胞，单侧疏松结缔组织中可见新生毛细血管和大量炎细胞浸润(见图3)。而经过干态处理的牛心包组织片在Wistar大鼠皮下植入后4周取材的组织学(HE)染色结果显示，牛心包组织中的纤维走向相对一致，连续性好，呈波浪状，心包组织中没有可见的炎细胞及炎细胞浸润，且心包组织内未见钙化灶(见图4)。由此可见，本发明所制备的牛心包组织具有较好的生物相容性，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0.625％(v/v)戊二醛溶液固定3天以上，得到经过戊二醛交联处理后的猪主动脉瓣膜。将处理后的猪主动脉瓣膜用20％(v/v)乙醇(国药集团化学试剂有限公司)的生理盐水在20℃下100rpm振荡清洗，清洗3～5次，每次清洗3～5分钟。之后将猪主动脉瓣膜浸没在含100％甘油(国药集团化学试剂有限公司)溶液的蓝盖瓶中，用锡箔纸完全包裹蓝盖瓶，20℃下浸没3小时。时间到后，将猪主动脉瓣膜在4℃下依次浸入到用锡箔纸完全包裹的、浓度分别为50％，60％，70％，75％，80％，85％(w/v)的果糖水溶液的蓝盖瓶中，每次浸没时间为30分钟。之后，在干燥的洁净环境中，再将猪主动脉瓣膜取出，放到覆膜纤维干燥剂上进行干燥。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。所制备的干态猪主动脉瓣膜组织的含水量为15.7±2.1％，再水化时间为4.85±0.78分钟，热皱缩温度为85.59±2.46℃，细胞毒性为1级，最大拉伸强度为11.9±4.3N。
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0.625％(v/v)戊二醛溶液固定3天以上，得到经过戊二醛交联处理的猪小肠粘膜下层组织。将处理后的猪小肠粘膜下层组织裁剪成小片(30mm×50mm)，用10％(v/v)异丙醇的生理盐水在25℃下100rpm振荡清洗猪小肠粘膜下层组织小片，清洗3～5次，每次清洗3～5分钟。之后将猪小肠粘膜下层组织浸没在含50％(v/v)的1,2,6-己三醇(上海晶纯生化科技股份有限公司)、30％(v/v)乙醇和20％(v/v)聚乙二醇400(上海晶纯生化科技股份有限公司)的混合醇溶液的蓝盖瓶中，用锡箔纸完全包裹蓝盖瓶，20℃下浸没2小时。时间到后，将猪小肠粘膜下层组织在20℃下依次浸入到用锡箔纸完全包裹的、浓度分别为30％，40％，50％，55％，60％，65％(w/v)的蔗糖水溶液的蓝盖瓶中，每次浸没时间为20分钟。之后，在干燥的洁净环境中，再将猪小肠粘膜下层组织取出，放到覆膜纤维干燥剂上进行干燥。最后，在干燥的洁净环境中，将干态的猪小肠粘膜下层组织放入透析袋中，密封，EO灭菌。所制备的干态猪小肠粘膜下层组织的含水量为16.5±1.6％，再水化时间为4.73±0.91分钟，热皱缩温度为87.34±1.15℃，细胞毒性为1级，最大拉伸强度为12.7±5.2N。
实施例4
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0.625％(v/v)戊二醛溶液固定3天以上。将固定后的猪心包裁剪成小片(30mm×50mm)，用15％(v/v)异丙醇的生理盐水在20℃下100rpm振荡清洗猪心包，清洗3～5次，每次清洗3～5分钟。之后将猪心包浸没在含75％的甘油和25％异丙醇的混合醇溶液的蓝盖瓶中，用锡箔纸完全包裹蓝盖瓶，20℃下浸没3小时。时间到后，将猪心包在20℃下依次浸入到用锡箔纸完全包裹的、浓度为30％，40％，50％，55％，60％，65％(w/v)的蔗糖水溶液的蓝 盖瓶中，每次浸没时间为10分钟。之后，在干燥的洁净环境中，再将猪心包取出，放到覆膜纤维干燥剂上进行干燥。最后，在干燥的洁净环境中，将干态的猪心包放入包装瓶中，密封，电子束辐照灭菌。所制备的干态猪心包组织的含水量为18.0±1.2％，再水化时间为5.15±0.36分钟，热皱缩温度为87.84±1.66℃，细胞毒性为1级，最大拉伸强度为14.2±5.8N。
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，在不脱离本发明的精神和范围内，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 (22)

  1. 一种干态动物源性胶原纤维组织材料的制备方法，其特征在于，包括如下步骤：
    (1)对经过交联剂交联处理后的动物源性胶原纤维组织材料进行清洗；
    (2)将清洗后的所述组织材料浸没在非水性醇溶液中进行脱水；
    (3)将经过非水性醇溶液脱水处理的所述组织材料依次浸没在不同浓度梯度的糖类水溶液中进行梯度脱水；
    (4)取出梯度脱水后的所述组织材料进行干燥；
    (5)将干燥后的所述组织材料密封包装后进行灭菌。
  2. 如权利要求1所述的制备方法，其特征在于，所述步骤(1)中动物源性胶原纤维组织材料为异种异体或同种异体的生物组织材料。
  3. 如权利要求2所述的制备方法，其特征在于，所述生物组织材料为心包、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(1)中的交联剂为戊二醛、京尼平、原花青素、炭化二亚胺中的一种或多种。
  5. 如权利要求1所述的制备方法，其特征在于，所述步骤(1)中的清洗过程如下：用体积百分比5％～30％(v/v)的异丙醇和/或乙醇的盐溶液在4～25℃下振荡清洗3～60分钟，振荡速率为50～150rpm。
  6. 如权利要求5所述的制备方法，其特征在于，所述盐溶液为生理盐水、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。
  7. 如权利要求1所述的制备方法，其特征在于，所述步骤(2)中的非水性醇溶液为分子量小于1000D的脂肪醇溶液。
  8. 如权利要求7所述的制备方法，其特征在于，所述非水性醇溶液为聚醚二醇和/或C2～C6脂肪醇溶液。
  9. 如权利要求8所述的制备方法，其特征在于，所述非水性醇溶液为聚 乙二醇、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、甘油、异丙醇和乙醇中的一种或多种。
  10. 如权利要求8所述的制备方法，其特征在于，所述非水性醇溶液中的组分包含聚乙二醇或甘油，且所述聚乙二醇或甘油的体积百分比为20％～90％；或者，所述非水性醇溶液中的组分包含聚乙二醇和甘油，且所述聚乙二醇与甘油的体积之和的百分比为20％～90％。
  11. 如权利要求9或10所述的制备方法，其特征在于，所述聚乙二醇的数均分子量为200～1000。
  12. 如权利要求1所述的制备方法，其特征在于，所述步骤(2)中非水性醇溶液的温度为20～37℃，所述组织材料在避光条件下浸没时间为30分钟～24小时。
  13. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中的糖类水溶液为具有吸水和保湿型的单糖、二糖、三糖、多糖或糖醇类糖水溶液。
  14. 如权利要求13所述的制备方法，其特征在于，所述糖类水溶液为果糖、蔗糖、海藻糖、非晶态的棉子糖、壳聚糖及壳聚糖改性的多糖、山梨糖醇或甘露糖醇水溶液。
  15. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中的梯度脱水过程如下：依次将经过非水性醇溶液脱水处理的所述组织材料浸入到不同浓度梯度的糖类水溶液，所述糖类水溶液温度为4～37℃，避光条件下每次浸没时间为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(3)中不同浓度梯度的糖类水溶液为浓度分别为30％，40％，50％，55％，60％，65％(w/v)的蔗糖水溶液；或者浓度分别为50％，60％，70％，75％，80％，85％(w/v)的果糖、海藻糖、非晶态的棉子糖、壳聚糖、壳聚糖改性的多糖、山梨糖醇或甘露糖醇的水溶液。
  17. 如权利要求1所述的制备方法，其特征在于，所述步骤(4)中取出 梯度脱水后的所述组织材料后放入纤维干燥剂进行干燥；或者在避光环境中控制温度20℃～37℃，晾放10分钟～24小时。
  18. 如权利要求17所述的制备方法，其特征在于，所述的纤维干燥剂为纤维干燥剂原片、折叠纤维干燥剂、袋片纤维干燥剂、柱状纤维干燥剂或覆膜纤维干燥剂。
  19. 如权利要求1所述的制备方法，其特征在于，所述的密封包装过程如下：在相对湿度小于30％的环境或惰性气体环境下将干燥后的所述组织材料放入到包装容器中进行密封包装。
  20. 如权利要求1所述的制备方法，其特征在于，所述的灭菌采用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，其特征在于，由权利要求1-20任一项所述的制备方法制取。
  22. 一种生物假体，其特征在于，由权利要求21所述的干态动物源性胶原纤维组织材料制成。
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