WO2013060103A1 - 一种处理动物源性胶原纤维材料的方法 - Google Patents

一种处理动物源性胶原纤维材料的方法 Download PDF

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WO2013060103A1
WO2013060103A1 PCT/CN2012/070482 CN2012070482W WO2013060103A1 WO 2013060103 A1 WO2013060103 A1 WO 2013060103A1 CN 2012070482 W CN2012070482 W CN 2012070482W WO 2013060103 A1 WO2013060103 A1 WO 2013060103A1
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solution
concentration
ethanol
tissue
animal
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PCT/CN2012/070482
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English (en)
French (fr)
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陈大凯
李�雨
房圆
田聪
董教明
周玲
刘翔
陈国明
乐承筠
罗七一
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上海微创医疗器械（集团）有限公司
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Priority to AU2012327785A priority Critical patent/AU2012327785A1/en
Priority to EP12842868.7A priority patent/EP2772274A4/en
Priority to BR112014009892A priority patent/BR112014009892A2/pt
Publication of WO2013060103A1 publication Critical patent/WO2013060103A1/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
    • A61L27/14—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
    • A61L27/3683—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
    • 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 the field of medical devices. More specifically, the present invention relates to a method of treating an animal-derived collagen-based material. Background technique
  • Animal-derived collagen fiber materials include tendons, corneas, heart valves, ligaments, skin and membranes, and are mainly composed of collagen fibers, elastic fibers, and proteoglycans.
  • the elastic fiber layer is distributed in the entire layer of the pericardium, and the collagen fibers have a wavy multi-layer structure.
  • the heart valve (heart valve) is composed of animal pericardial components, which are mainly divided into a serosa layer, a fibrous layer and an outer pericardial connective tissue layer, which is a kind of animal-derived collagen fiber material.
  • Bio-heart valve is a biological material used for the treatment of heart diseases. Compared with mechanical valves, it has the advantages of no long-term anticoagulation and good hemodynamic performance.
  • the first is glutaraldehyde fixation and chemical modification.
  • Glutaraldehyde treatment can crosslink collagen fibers to increase tissue stability, reduce immunogenicity, and help to increase its mechanical properties and durability in vitro. However, it also loses a large amount of soluble protein, its surface exposed amino acids. The residue changes, the phosphate bond of collagen is exposed, providing a chemical environment similar to stimulating calcification [Golomb G; Ezra V Govalent binding of protamine by glutaraldehyde to biopros the ic tissue: characterization and ant icalcif icat ion effect 1992 (01)].
  • the second method is the decellularized biological valve.
  • the cells of the original tissue are removed, and a matrix is constructed so that cells implanted in the body can be grown thereon.
  • Specific methods are: 1) Decellularization (killing cells by osmotic shock and nuclease treatment) [Goldstein, US Pat. Nos.
  • the autologous thin 3 packs are planted, so that the bio-valve area i or re-endothelialization [Goldstein, US Pat. No. 7318998] D is not ideal for clinical application.
  • the first case was applied to clinical surgery in Europe using the decellularized pig tissue engineering valve SynergrafteTM (Cryolife Inc., USA), but tearing, calcification and severe degeneration of the valve occurred shortly after surgery [Simon P. Kasimir MT. Seebacher G et al.
  • Polyethylene glycol is a low toxicity, non-immunogenic compound with good biocompatibility and does not degrade in vivo to produce toxic derivatives with little effect on the biological properties of the modified substance. It has been found that polyethylene glycol (PEG) acts on implants to reduce the immune response of the receptor [Heart Preservation Solution Containing Polyethylene Glycol: An Immunosuppressive Effect" by Col 1 ins, et al. in Lancet,
  • mice implanted with PEG-treated mice increased from 9.6 days to 11.9 days [ "The Imtnunosuppres sive Effect of Polyethyene Glycol in a Flush Solution for Rat Liver Transplantation "by Tokunaga, et al. in Transplantation, 54: 756 8 (1992)].
  • Histocompatibility antigens have also been found to be a major determinant of transplant rejection.
  • the human immune system recognizes foreign tissues, produces inflammatory cells such as macrophages, secretes microparticles containing enzymes, and digests foreign matter.
  • the present invention provides a method of treating an animal-derived collagen tissue material that can be implanted into a recipient without an immune inflammatory response.
  • the present invention uses hydrogen peroxide as an oxidizing agent.
  • the oxidant naturally produced during the inflammatory reaction of the human body is also hydrogen peroxide, which reacts with various groups to oxidize it, such as aldehyde groups, amino groups, lipids and the like. In the oxidation reaction, it is necessary to protect the tissue fibers from damage by denaturation.
  • the present invention relates to a method of treating an animal-derived collagen fiber material, characterized in that the animal-derived collagen fiber material is treated with a salt solution containing polyethylene glycol and an oxidizing agent. 5 ⁇ / L - 4. 5mo l / L.
  • the concentration of the salt solution is at least 2. 5mo l / L, preferably 2. 5mo l / L - 4. 5mo l / L.
  • the oxidizing agent is preferably hydrogen peroxide.
  • the animal-derived collagen fibrous material is a tendon, a cornea, a heart valve, a ligament, a skin and a septum, preferably a biological valve.
  • the method of the invention comprises the following steps:
  • tissue material After the tissue material is taken out from the animal body, it is trimmed in physiological saline, and then immersed in 0-1 (TC stable solution, the stable solution is physiological saline or 30% - 70% (preferably 40% - 60%, more preferably 50%) Ethanol, soaking time should not exceed when using normal saline
  • the soaking time should not exceed 30 days when using ethanol
  • the tissue is then treated with solution one.
  • the composition of the solution is as follows: polyethylene glycol, molecular weight 2000-20000D, concentration 1%-15%; sodium chloride, concentration 1, 5mol/L-4.5mol/L; phosphate buffer , concentration 0.02- 0. lmol / L, pH 6.5-7; oxidant, concentration 0.1% -2%; treatment time 2 - 2400 hours, temperature 0 - 1 (TC;
  • the method of the invention further comprises:
  • the tissue is treated with the solution 2.
  • the two components of the solution are: 30% - 70% (preferably 40% - 60%, more preferably 50%) ethanol; anti-inflammatory drug, concentration 10-200 mg / L;
  • the tissue is immersed in solution three, the three components of the solution are: 30% - 70 (preferably 40% - 60%, more preferably 50%) ethanol; anticoagulant, concentration 100-1000 IU / ml;
  • the tissue is washed with physiological saline, immersed in 30%-70% (preferably 4 «-60%, more preferably 50%) ethanol, and stored at 0-6 °C.
  • the method of the present invention further comprises: After solution-treatment and aqueous ethanol washing, immersed in solution four, the four components of the solution are: 30% - 70% (preferably 40% - 60%, more preferably 50 ° /.) ethanol; anti-inflammatory drugs, concentration 1 0- 200mg / L; anticoagulant, concentration 100-1000 IU / ml;
  • the tissue is washed with physiological saline, immersed in 30%-70% (preferably 40%-60%, more preferably 50%) ethanol, and stored at 0-6 °C.
  • the anti-inflammatory agent is selected from the group consisting of indomethacin, dexamethasone acetate, dexamethasone, rapamycin, prednisone, prednisone acetate, esculinol acetate, pico test Ibuprofen, ibuprofen and butyl hydroxy acid, and anticoagulants are selected from heparin, enoxaparin and hirudin.
  • Figure 1 is a flow chart of the treatment of animal-derived collagen tissue material.
  • Fig. 2 is a comparison of the calcium content of mice treated with the bovine pericardial bioprosthesis treated with glutaraldehyde in the first embodiment, the first embodiment and the third embodiment of the present invention. detailed description
  • the artificial biological valve treated in step 1 is placed in polyethylene glycol, concentration 5%, molecular weight 2QQG D; sodium chloride, concentration 2.5mol/L; phosphate buffer, concentration 0.04mol/L, pH 7; Hydrogen peroxide, 1% solution, treatment time 80 hours.
  • Example 2
  • the artificial biological valve treated in step 1 is placed in polyethylene glycol, concentration 5%, molecular weight 6000 D; sodium chloride, concentration 2.5 mol/L; phosphate buffer, concentration 0.02 mol/L, pH 7; Hydrogen peroxide, in a solution of 1% concentration, the treatment time is 90 hours.
  • pH of the artificial biological valve treated in step 1 was placed in polyethylene glycol, concentration 15%, molecular weight 80QG D; sodium chloride, concentration 4.5mol / L; phosphate buffer, concentration 0. lmol / L, pH Value 7; hydrogen peroxide, 2% solution, treatment time 96 hours.
  • step 3 Place the artificial biological valve treated in step 2 in 50% ethanol; indomethacin, in a solution with a concentration of 200 mg/L, for a treatment time of 30 hours.
  • the artificial biological valve treated in step 3 was placed in 50% ethanol; enoxaparin, in a solution at a concentration of 1000 IU/ml, for 24 hours.
  • Example 4 1.
  • the artificial biological valve was immersed in Q. 625% glutaraldehyde solution for 10 days to obtain an artificial biological valve (bovine pericardial biological valve) treated with glutaraldehyde as a control.
  • Sample implantation The artificial biological valve of Example 1-3 and the control biological valve were implanted into the rat for 35 days and taken out.
  • Sample preparation The biological valve is removed, rinsed to remove blood, and placed in an oven, 90.
  • Standard preparation Precisely measure the standard solution of calcium in a volumetric flask, add an appropriate amount of concentrated nitric acid diluted in a volume ratio of 1:1, add the same amount of hydrogen peroxide as the sample, and then dilute to the mark with water. kind.
  • the calcium content of the artificial biological valve is obtained by the atomic absorption spectrophotometer (AAS) for the change of the absorbance during the combustion process of the artificial biological valve decomposition liquid.
  • AAS atomic absorption spectrophotometer

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Abstract

一种处理动物源性胶原纤维材料的方法，包括使用含有聚乙二醇和氧化剂的盐盐溶液对动物源性胶原纤维材料进行处理。所述方法处理后的动物源性胶原纤维材料植入受体后，不发生免疫炎症反应。

Description

一种处理动物源性胶原纤维材料的方法 技术领域
本发明涉及医疗器械领域。 更具体而言， 本发明涉及一种处 理动物源性胶原紆维材料的方法。 背景技术
动物源性胶原纤维材料包括肌腱、 角膜、 心瓣、 韧带、 皮肤 和隔膜等， 主要由胶原纤维、 弹性纤维和蛋白聚糖组成。 弹性纤 维层网状分布于心包全层， 胶原纤维呈波浪状多层次结构。 心脏 瓣膜 （心瓣） 是由动物心包組分所组成， 主要分为浆膜层、 纤维 层和外心包结締组织层， 它是动物源性胶原纤维材料的一种。 生物心脏瓣膜是用于心脏疾病治疗的生物材料， 与机械瓣膜 相比， 具有不需长期抗凝、 血流动力学性能良好等优点， 尽管其 使用寿命仍然较低， 但它的临床应用地位无法用机械瓣代替。 目 前国外生物瓣使用比例已经超过机械瓣。 但由于移植中存在免疫 反应， 生物瓣需要事先经过特殊处理。 现有技术对异种生物瓣的处理有以下几种方法：
首先是戊二醛固定与化学改性。 戊二醛处理能使胶原纤维发 生交联从而增加组织稳定性， 降低免疫原性， 有助于增加其体外 的力学性能与耐久性； 但同时也丢失了大量的可溶性蛋白， 其表 面暴露的氨基酸残基发生变化， 胶原的磷酸键裸露， 提供了类似 激发钙化的化学环境 [Golomb G; Ezra V Govalent binding of protamine by glutaraldehyde to biopros thet ic tissue: characterization and ant icalcif icat ion effect 1992 (01)]。 因此， 在戊二醛固定的基础上， 各种改性技术被发展应用： 清洁 剂或表面改性剂的处理， 共价结合二磷酸盐类， α油酸防钙化处 理， 甲苯胺蓝处理等。 这些方法延緩了瓣膜组织疲劳和钙化的发 生， 但并没有从根本上解决问题。 第二种方法为去细胞生物瓣。 去除原组织的细胞， 构造一个 基质，使得植入人体后受体的细胞能在其上生长。具体方法有： 1 ) 脱细胞（通过氐渗休克和核酸酶处理杀死细胞） [Goldstein, U.S. Pat. Nos. 5613982; 5632778; 5899936和 5843182]； 2) 控制下 的细胞自我分解 [Jaffe, U.S. Pat. Nos. 5843180; 5843181 和 5720777]； 3)辐射 [Schinstein, U.S. Pat. Nos. 5795790; 8843431; 5843717 和 5935849] [Badylak, U.S. Pat. No. 6126686]； 4) 酸处理 [Abraham, U.S. Pat. No. 5993833] 0 移植入人体后， 在 体内环境下人体细胞在其上分化为内皮细胞， 最终材料将重塑为 类似天然的组织。 第三种方法为体外内皮化。 瓣膜材料脱细胞后种植、 培养自 体细 3包，使生物瓣棵露的区 i或再内皮化 [Goldstein, U.S. Pat. No. 7318998] D此种生物瓣在临床初步应用的效果并不理想。 2003年， 第 1例用去细胞猪生物组织工程瓣 Synergrafte™ (Cryolife Inc. , USA)在欧洲应用于临床手术， 但术后短期内即出现了瓣膜的撕裂、 钙化和严重的变性 [Simon P. Kasimir MT. Seebacher G et al.
Early failure of the tissue engineered porcine heart valve SYNERGRAFT in pediatric patients European Journal of Cardio— Thoracic Surgery. Volume 23, Issue 6, June 2003, Pages 1002-1006] 0 由于还不清楚干细胞分化的分子机制， 定向诱导分化过程也 不能被完全控制， 因而生物瓣体外内皮化还有很多问题需要解决。 针对上述诸多缺陷，生物瓣膜至今尚无令人满意的解决方案， 仍有进一步改进的余地。 本领域技术人员知晓， 免疫反应的主要原因是供体与受体细 胞表面分子的差异。 如果植入物含有供体活细胞， 免疫反应会被 增强； 若植入物不含活细胞， 其残留的蛋白等物质仍具有免疫原 性。 为了去除这些物盾， 必须使用强效的化学处理手段， 以保证 所有这些物质都被除去。 而在这过程中组织基质同样也遭到了破 坏。 聚乙二醇（PBG)是一种低毒性、 无免疫原性的化合物， 具有 良好的生物相容性， 不会在体内降解产生有毒的衍生物， 几乎不 影响被修饰物质的生物学性质。 人们已经发现聚乙二醇（PEG)作 用于植入物， 能够降低受体的免疫反应〖 "Heart Preservation Solution Containing Polyethylene Glycol: An Immunosuppressive Effect" by Col 1 ins, et al. in Lancet,
338: 390 (1991)]。 Tokunaga等人进行的小鼠肝脏移植实验中， 植 入组织经 PEG 处理的实验組小鼠平均存活时间由 9.6 天提高到 11.9天 [ "The Imtnunosuppres sive Effect of Polyethyene Glycol in a Flush Solution for Rat Liver Transplantation" by Tokunaga, et al. in Transplantation, 54: 756 8 (1992)]。 亦已发现组织相容性抗原是移植排斥产生的主要决定因素。 当接受来自不同种或不同个体的植入物时， 人体免疫系统识别出 外来組织， 产生炎症细胞如巨噬细胞， 分泌含有酶的微粒， 消化 外来物。 而消化的过程又会产生更多的炎症细胞， 直到外来物被 去除。 此消化过程是一种氧化反应， 因此炎症反应与植入物可被 氧化的程度有关。 然而，现有技术并没有教导 PEG与氧化剂组合处理生物瓣膜， 降低炎症反应的方法。 发明内容
本发明提供一种处理动物源胶原组织材料的方法， 使之能植 入受体， 而不发生免疫炎症反应。 为了降低植入物可被氧化的程度， 避免炎症反应的发生， 本 发明使用过氧化氢作为氧化剂。 人体的炎症反应过程中自然产生 的氧化剂也是过氧化氢， 它可与多种基团反应使之被氧化， 如醛 基、 氨基、 脂质等。 在氧化反应中， 需要保护组织纤维， 以免变 性而遭破坏。 人们已经知道在高浓度盐溶液中， 胶原分子及动物 纤維能维持稳定， 因此本发明氧化反应在高浓度盐溶液中进行， 从而保护組织纤维。 结締组织主要由胶原、 弹性纤维、 胶原蛋白、 蛋白聚糖、 脂 质、 核酸、 可溶性小分子、 无机物、 水等组成。 当细胞死亡后， 小分子被释放出来， 而蛋白质、 蛋白聚糖、 核酸等大分子不易除 去。 高浓度盐溶液能析出这些大分子， 同时破坏蛋白聚糖与核酸 之间的相互作用， 使它们不再粘附于胶原。 在去除非胶原物质及氧化反应过程中， 为了使胶原纤维保持 完整， 在溶液中加入聚乙二醇以使胶原纤维不被溶解。 具体而言， 本发明涉及一种处理动物源性胶原纤维材料的方 法， 其特征在于， 用含有聚乙二醇和氧化剂的盐溶液对动物源性 胶原纤维材料进行处理。 根据本发明， 盐溶液的浓度至少为 2. 5mo l /L , 优选为 2. 5mo l /L— 4. 5mo l /L。 根据本发明， 氧化剂优选为过氧化氢。 根据本发明， 动物源性胶原纤维材料为肌腱、 角膜、 心瓣、 韧带、 皮肤和隔膜， 优选为生物瓣膜。 本发明方法包括下列步骤：
将組织材料从动物体内取出后， 在生理盐水中修剪， 然后浸 泡于 0- 1(TC稳定液中， 稳定液为生理盐水或 30%- 70% (优选 40%-60%, 更优选 50%) 乙醇， 使用生理盐水时浸泡时间不应超过
48小时， 使用乙醇时浸泡时间不应超过 30天；
然后用溶液一处理组织， 溶液一成分如下： 聚乙二醇， 分子 量 2000-20000D,浓度 1%-15%；氯化钠，浓度 1、 5mol/L-4.5mol/L; 磷酸盐緩冲液， 浓度 0.02- 0. lmol/L, pH值 6.5-7; 氧化剂， 浓度 0.1%-2%； 处理时间 2- 2400小时， 温度 0- 1(TC;
从溶液一中取出组织后， 用低浓度（例如 , 60%以下） 乙醇水 溶液清洗， 洗去溶液一的残余物。 本发明方法进一步包括：
在溶液一处理和乙醇水溶液清洗后， 再用溶液二处理组织， 溶液二成分为： 30%- 70% (优选 40%- 60%， 更优选 50% ) 乙醇； 消 炎药物， 浓度 10- 200mg/L;
然后任选地，将组织浸泡于溶液三中，溶液三成分为： 30%-70 (优选 40%-60%, 更优选 50%) 乙醇； 抗凝血药， 浓度 100-1000 IU/ml;
最后用生理盐水冲洗组织， 浸没于 30%-70% (优选 4«- 60%, 更优选 50%) 乙醇中， 0- 6°C保存。 或者， 本发明方法进一步包括： 在溶液一处理和乙醇水溶液清洗后， 浸泡于溶液四中， 溶液 四成分为： 30%- 70% (优选 40%- 60% , 更优选 50°/。） 乙醇； 消炎药 物， 浓度 1 0- 200mg/L; 抗凝血药， 浓度 100- 1000 IU/ml ;
最后用生理盐水冲洗組织， 浸没于 30%-70% (优选 40%-60%， 更优选 50% ) 乙醇中， 0- 6 °C保存。 在本发明的优选方案中， 消炎药选自吲哚美辛， 醋酸地塞米 松， 地塞米松， 雷帕霉素， 泼尼松， 醋酸泼尼松， 醋柳酸娠烯醇 酮， 皮考布洛芬， 布洛芬和丁苯羟酸， 而抗凝血药选自肝磷脂、 依诺肝素和水蛭素。 除心瓣之外， 其他动物源性胶原纤维材料， 如肌腱、 角膜、 韧带、 皮肤和隔膜等， 其主要成分也是由胶原纤维、 弹性纤维和 蛋白聚糖组成， 所以本领域技术人员可以预期， 本发明采用的对 于瓣膜材料的处理方法同样适用于这些动物源性胶原纤维材料。 附图说明
为了更清楚地描述本发明的技术方案， 下面将结合附图作简 要介绍。 显而易见， 这些附图仅是本申请记载的一些具体实施方 式。 本发明方法包括但不限于这些附图。 图 1为动物源胶原组织材料的处理流程图。
图 2采用本发明实施例 1、 实施例 1和实施例 3与戊二醛处 理后的牛心包生物瓣膜植入小鼠 35天后的钙含量比较。 具体实施方式
为了进一步理解本发明， 下面将结合实施例对本发明的优选 方案进行描述。 这些描述只是举例说明本发明方法的特征和优点， 而非限制本发明的保护范围。 实施例 1
1. 将人工生物瓣膜在 50%乙醇溶液中浸泡 10天。
2. 将步骤 1处理过的人工生物瓣膜放入聚乙二醇， 浓度 5%, 分子量 2QQG D; 氯化钠， 浓度 2.5mol/L; 磷酸盐緩冲液， 浓度 0.04mol/L, pH值 7; 过氧化氢， 浓度 1%的溶液中， 处理时间 80 小时。 实施例 2
1. 将人工生物瓣膜在 50%乙醇溶液中浸泡 20天。
2. 将步骤 1处理过的人工生物瓣膜放入聚乙二醇， 浓度 5%， 分子量 6000 D; 氯化钠， 浓度 2.5mol/L; 磷酸盐緩冲液， 浓度 0.02mol/L, pH值 7; 过氧化氢， 浓度 1%的溶液中， 处理时间为 90小时。
3. 将步骤 2处理过的人工生物瓣膜放入 50% 乙醇； 吲哚美 辛， 浓度 100mg/L的溶液中， 处理时间为 30小时。 实施例 3
1. 将人工生物瓣膜在 50%乙醇 液中浸泡 30天。
2. 将步骤 1处理过的人工生物瓣膜放入聚乙二醇，浓度 15%， 分子量 80QG D; 氯化钠， 浓度 4.5mol/L; 磷酸盐緩冲液， 浓度 0. lmol/L, pH值 7; 过氧化氢， 浓度 2%的溶液中， 处理时间为 96 小时。
3. 将步骤 2处理过的人工生物瓣膜放入 50%乙醇；吲哚美辛， 浓度 200mg/L的溶液中， 处理时间为 30小时。
4. 将步骤 3处理过的人工生物瓣膜放入 50% 乙醇； 依诺肝 素， 浓度 1000 IU/ml的溶液中， 处理时间为 24小时。 实施例 4 1. 将人工生物瓣膜在 Q. 625%戊二醛溶液中浸泡 1 0天，得到 戊二醛处理后的人工生物瓣膜 （牛心包生物瓣膜） ， 作为对照。
2. 样品植入：将实施例 1-3的人工生物瓣膜和对照生物瓣膜 植入大鼠皮下 35天后取出。
3. 样品配制： 将生物瓣膜取出， 漂洗去除血液， 然后置于烘 箱中， 90。C X 40h烘干， 标准精确称量干重后用体积比 1 : 1稀释的 浓硝酸加热分解， 冷却后过氧化氢充分氧化， 然后用水定容至刻 度， 待进样。
4. 标准品配制： 精密量取钙的标准溶液于容量瓶中，加入体 积比 1 : 1 稀释的浓硝酸适量， 再加入与样品等量的过氧化氢， 然 后用水定容至刻度， 待进样。
5. 钙含量的测定方法： 通过原子吸收分光光度计 （AAS )对 于人工生物瓣膜分解液燃烧过程中的吸光值变化得出人工生物瓣 的钙含量。
AAS参数:
Figure imgf000011_0001
钙含量的具体数值参见图 2
以上实施例的说明只是用于帮助理解本发明的核心思想。 应 当指出， 对于本领域的普通技术人员而言， 在不脱离本发明原理 的前提下， 还可以对本发明方法进行若干改进和修饰， 但这些改 进和修饰也落入本发明权利要求请求保护的范围内。

Claims

权 利 要 求 书
1. 一种处理动物源性胶原纤维材料的方法， 其特征在于， 用 含有聚乙二醇和氧化剂的盐溶液对动物源性胶原纤维材料进行处 理。
2. 权利要求 1 所述的方法， 其中盐溶液的浓度至少为 2.5mol/L。
3. 权利要求 1或 2所述的方法， 其中氧化剂为过氧化氢。
4. 前述权利要求任一项所述的方法，其中动物源性胶原纤维 材料为肌腱、 角膜、 心瓣、 韧带、 皮肤或隔膜。
5. 权利要求 4所述的方法，其中动物源性胶原纤维材料为生 物瓣膜。
6. 前述权利要求任一项所述的方法， 其包括下列步骤： 将组织材料从动物体内取出后， 在生理盐水中修剪， 然后浸 泡于 0-10°C稳定液中， 稳定液为生理盐水或 30%-70%乙醇， 使用 生理盐水时浸泡时间不应超过 48小时，使用乙醇时浸泡时间不应 超过 30天；
然后用溶液一处理组织， 溶液一成分如下： 聚乙二醇， 分子 量 2000- 20000D，浓度 1%-15%； 氯化钠，浓度 2.5mol/L- 4, 5mol/L; 磷酸盐緩冲液， 浓度 0.02- 0. lmol/L, pH值 6.5 7; 氧化剂， 浓度
0.1%-2%； 处理时间 2- 2400小时， 温度 0- 10°C;
从溶液一中取出组织后， 用低浓度乙醇水溶液清洗， 洗去溶 液一的残余物。
7. 权利要求 6所述的方法， 其进一步包括： 在溶液一处理和乙醇水溶液清洗后 , 再用溶液二处理组织， 溶液二成分为： 30%- 70°/。乙醇； 消炎药物， 浓度 10-2 00nig/L; 然后任选地，将组织浸泡于溶液三中，溶液三成分为： 30°/。- 70% 乙醇； 抗凝血药； 浓度 100-1 000 IU/ml ;
最后用生理盐水冲洗组织， 浸没于 30%- 70%乙醇中， 0-6 °C保 存。
8. 权利要求 6所述的方法， 其进一步包括：
在溶液一处理和乙醇水溶液清洗后， 浸泡于溶液四中， 溶液 四成分为： 30%-70%乙醇； 消炎药物， 浓度 1 0-200mg/L; 抗凝血药， 浓度 100-1000 IU/ml ;
最后用生理盐水冲洗组织， 浸没于 30¾- 70%乙醇中， 0- 6 °C保 存。
9. 权利要求 7或 8所述的方法， 其中消炎药选自吲哚美辛， 醋酸地塞米松， 地塞米松， 雷帕霉素， 泼尼松， 醋酸泼尼松， 醋 柳酸娠烯醇酮， 皮考布洛芬， 布洛芬和丁苯羟酸， 而抗凝血药选 自肝磷脂、 依诺肝素和水蛭素。
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TOKUNAGA ET AL.: "The Immunosuppressive Effect of Polyethylene Glycol in a Flush Solution for Rat Liver Transplantation", TRANSPLANTATION, vol. 54, 1992, pages 756 8

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BR112014009892A2 (pt) 2017-04-18
EP2772274A4 (en) 2015-06-17
CN102350008A (zh) 2012-02-15
EP2772274A1 (en) 2014-09-03
AU2012327785A1 (en) 2014-06-19

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