WO2016172894A1 - 一种灭活胶原材料及其制备方法 - Google Patents

一种灭活胶原材料及其制备方法 Download PDF

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WO2016172894A1
WO2016172894A1 PCT/CN2015/077898 CN2015077898W WO2016172894A1 WO 2016172894 A1 WO2016172894 A1 WO 2016172894A1 CN 2015077898 W CN2015077898 W CN 2015077898W WO 2016172894 A1 WO2016172894 A1 WO 2016172894A1
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collagen material
inactivated
collagen
preparing
inactivation
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PCT/CN2015/077898
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English (en)
French (fr)
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杨军
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上海欣吉特生物科技有限公司
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Priority to PCT/CN2015/077898 priority Critical patent/WO2016172894A1/zh
Publication of WO2016172894A1 publication Critical patent/WO2016172894A1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the invention relates to an inactivating material prepared by using natural collagen as a raw material and a preparation method thereof, in particular to an inactivating material prepared by using natural collagen in animal tissues and a preparation method thereof.
  • Collagen or collagen is a biopolymer synthesized by animal cells. It is widely found in animal bones, cartilage, tendons, skin and other connective tissues. It accounts for about one-third of total mammalian protein and is important for human body. Extracellular matrix component. Collagen is uniquely used in the field of tissue engineering materials. It has a wide range of sources, low cost, good biocompatibility, biodegradability and low antigenicity. It has good hydrophilicity and its porous structure is good for cell adhesion.
  • an extracellular matrix component As an extracellular matrix component, it has an inducing differentiation effect on cells, and can be used as a supporting substance for tissues, and has an important influence on the normal function of cells, tissues, and organs, and wound repair, and is manufactured into respective wound healing materials, scaffold materials, and the like.
  • the field of manufacture of device products has irreplaceable advantages and a wide range of applications.
  • collagen materials have many natural advantages, most of the collagen is derived from animals. As its use in clinical medical products is increasingly widespread, the safety risks of viral transmission and immunogenicity are increasing. Therefore, the risk of the virus from the source organization, the risk of carrying infectious agents, and the potential harm to the human body have caused widespread concern. In order to ensure the safety of the final medical product, animal-derived raw materials need to be strictly controlled to increase effective virus inactivation or removal measures in the production process.
  • virus inactivation methods include pasteurization, dry heat inactivation, peracetic acid-ethanol inactivation, ethanol inactivation, radiation inactivation, NaOH Inactivation method, etc.
  • Pasteurization is one of the wet heat inactivation methods. It is a recognized method of virus inactivation at home and abroad. It has been successfully applied in human albumin products for decades, and the inactivation conditions are perfect. This inactivation method inactivates the lipid envelope and a portion of the non-lipid enveloped virus. Allogeneic implantable medical devices can be used for virus inactivation after adequate cleaning of blood and bone marrow components. The homogeneity and inactivation time of the temperature distribution should be considered when using this method.
  • the dry heat inactivation method is mainly used for virus inactivation of freeze-dried products.
  • the virus inactivation effect of this method has been confirmed by laboratory verification and clinical application, and can inactivate HIV.
  • the advantage of the dry heat inactivation method is that it can be used for allogeneic implantable medical devices after lyophilization. The uniformity of temperature distribution, lyophilization process and product moisture residue should be considered when using this method.
  • Peracetic acid has a strong ability to inactivate viruses; ethanol reduces the surface tension of the solution and helps the disinfectant penetrate completely into the allogeneic implantable medical device.
  • peracetic acid The ethanol inactivation method is a reliable method of virus inactivation, inactivated poliovirus (PV), HIV, pseudorabies virus (PRV); bovine viral diarrhea virus (BVDV); porcine parvovirus ( A variety of viruses, such as PPV, have been shown to be effective in laboratory and clinical trials for the inactivation of allogeneic implantable medical devices.
  • the safe peracetic acid residue limit for human use should be strictly controlled when using this method.
  • Ethanol is the most commonly used surface disinfectant in the clinic. This method is suitable for most enveloped viruses such as herpes simplex virus, HIV And so on.
  • the virus used for allogeneic bone is inactivated, the blood and bone marrow components should be thoroughly cleaned; and because ethanol has strong volatility, soaking should be used to prevent high temperature and open flame. Since different methods have different effects on the product, the actual use depends on the nature of the material and the type of virus carried by the material.
  • the main advantages of the ray irradiation inactivation method include high inactivation efficiency, strong penetrability, easy dose control, no harmful substances, and no obvious temperature rise.
  • This method inactivates the virus primarily by disrupting the nucleic acid. Since the virus has different radiation resistance in different media, the method should be used to remove the host tissues and cells in the product when the virus is inactivated in the allogeneic implantable medical device. For example, the allogeneic bone should be thoroughly cleaned. Blood and bone marrow components. Irradiation dose should be determined based on the characteristics of the product. Irradiation dose distribution and inactivation time should be considered when using this method.
  • Sodium hydroxide has been considered to be effective in removing proteins and nucleic acids. It also inactivates most viruses, bacteria, yeast, fungi and endotoxins.
  • One of the great benefits of using sodium hydroxide is that it is efficient, low cost, and easy to detect, remove, and handle.
  • Sodium hydroxide can be effectively inactivated BSE virus, which is quite resistant to most treatments, including 360 Dry at °C for one hour.
  • due to the destruction of the peptide bond in natural collagen by sodium hydroxide it will hydrolyze and inactivate, and will also greatly reduce the mechanical properties of collagen.
  • virus inactivation methods described above will cause damage to the natural structure and function of collagen to varying degrees.
  • collagen materials are also susceptible to degradation and poor mechanical properties. In practice, they are often modified to improve their mechanical properties.
  • modification methods of collagen mainly include physical crosslinking, chemical crosslinking, and modification by blending with other polymer materials. Physical methods usually include ultraviolet radiation, severe dehydration, gamma ray irradiation, and thermal crosslinking.
  • the advantage is that exogenous toxic chemicals can be prevented from entering the collagen.
  • the disadvantage is that the degree of collagen cross-linking is low and it is difficult to obtain uniform cross-linking.
  • Chemical methods usually use cross-linking with chemical reagents, modification of side chains, and immobilization of physiological active substances.
  • the advantage is that the degree of cross-linking is higher than that of physical methods, and uniform cross-linking can be obtained, and each of the collagen is regulated and controlled.
  • the effect of uniformity and goodness is that the introduction of exogenous toxic reagents and the removal of residual reagents are difficult to remove.
  • Chemical cross-linking agents can be divided into two categories according to their functions: the first type is a polyfunctional cross-linking agent having a bifunctional group, for example, a diisocyanate and an epoxy compound. (BDDGE, Jingni Equality) Such reagents form an amine bridge between two adjacent peptide chains; a second type of crosslinker can activate a carboxylic acid on a glutamic acid or aspartic acid residue to allow it to interact with another peptide chain The amine group reaction forms an amide bond providing a crosslink bond.
  • the cross-linking agent can be divided into two types according to the cross-linking bond: one is intramolecular cross-linking, which is a cross-linking bond formed between two peptide chains in the same helix, and such cross-linking bonds mainly affect The properties of the cross-linked product such as denaturation temperature and tensile strength; the other is intermolecular cross-linking, which is a cross-linking bond formed in a peptide chain between two adjacent helices.
  • Such crosslinks primarily affect the swellability and surface extensibility of the crosslinked product.
  • Crosslinking can also occur between two adjacent microfibers when the distance between the two microfibers is less than the length of the crosslinker molecules.
  • Thermal shrinkage temperature, tensile strength and degradation strength are important indicators for evaluating the cross-linking properties of biomaterials. The higher the thermal shrinkage temperature and tensile strength test values, the stronger the mechanical properties.
  • Crosslinking treatment of biological materials by various cross-linking agents will increase their heat shrinkage temperature, tensile strength and degradation resistance, while the increase of heat shrinkage temperature, tensile strength and degradation resistance indicates that the crosslinking treatment is improved.
  • the mechanical and biological stability of the product The factors determining the degree of intermolecular cross-linking reaction are the conformation of the protein itself and the nature of the cross-linking agent used.
  • the cross-linking agents of different properties have a great difference in the degree of cross-linking of collagen.
  • the extracted or natural collagen is usually inactivated first, and the inactivation process often causes different degrees of damage to the chemical, biological properties and structure of the collagen, and affects the function of the collagen. Therefore, the present invention seeks a method for effectively inactivating collagen while retaining the three-dimensional structure, biological activity and mechanical properties of natural collagen, thereby making the prepared collagen material safe and effective.
  • the object of the present invention is to provide a safe and effective collagen material by providing a simple, efficient and cost-effective method for preparing an inactivated collagen material. More specifically, the main object of the present invention is to solve the problem of causing damage to the three-dimensional structure of natural collagen during sodium hydroxide inactivation or/and electron beam irradiation inactivation resulting in deterioration of its mechanical and biological properties.
  • the technical solution adopted by the present invention is: a preparation method of inactivating collagen material: firstly performing natural chemical crosslinking on natural collagen, and then performing sodium hydroxide inactivation or/and electron beam irradiation Inactivated treatment; this can remove potential viral threats, further reduce immunogenicity, and retain the three-dimensional structure, biological properties and mechanical properties of natural collagen.
  • the inactivated collagen material prepared by the above method can be implanted as a medical collagen material, and the cells around the damaged part enter into the growth and promote the healing of the damaged tissue.
  • the method for preparing the inactivated collagen material of the present invention comprises the following specific steps:
  • the collagen material treated in step 1) is completely immersed in the cross-linking agent to fix the collagen; the cross-linking agent chemically reacts with the active amino group and carboxyl group between the collagen molecules to reduce the collagen substrate. Immunogenicity and increase the mechanical strength of the substrate itself;
  • step 3 inactivation: the collagen material treated in step 2) is completely immersed in a NaOH solution at room temperature (25 ° C) for inactivation treatment, or / and electron beam irradiation inactivation treatment;
  • the physical method in the step 1) includes, for example, hand tearing, cutting with scissors, using a tweezers clip, etc.;
  • the collagen material in the step 1) is a tissue containing more collagen such as pericardium, skin, tendon or the like of an animal such as pig or cow.
  • the crosslinking agent in the step 2) may be a compound such as an aldehyde, a carbon diamine, an epoxy or a cyanate, preferably glutaraldehyde, ethylene glycol diglycidyl ether (Denacol) or carbon.
  • Diethylene diamine hydrochloride (EDC) when the crosslinking agent is glutaraldehyde, an aqueous solution of glutaraldehyde is preferred, and the optimum concentration is 0.5% to 1% (w/v).
  • the crosslinking time is not less than 1 hour; when ethylene glycol diglycidyl ether is used as the crosslinking agent, the crosslinking time is When not less than 1 day or carbon diamine hydrochloride as a crosslinking agent, the crosslinking time is not less than 0.5 h.
  • the concentration of NaOH in step 3) is 1-2 mol/L and the soaking time is 0.5-1 hour.
  • the irradiation dose inactivated by electron beam irradiation in step 3) is 15-40 kGy.
  • the specific operation of the washing to neutral in step 4) is to sequentially rinse to neutral with buffer, physiological saline and water for injection.
  • the above buffer is CBS (i.e., sodium carbonate-sodium bicarbonate buffer solution) or / and PBS (phosphate buffered saline solution).
  • CBS sodium carbonate-sodium bicarbonate buffer solution
  • PBS phosphate buffered saline solution
  • the final properties of the prepared collagen material can be controlled by controlling the concentration of the crosslinking agent and the crosslinking time. Theoretically, the greater the concentration of the crosslinking agent, the longer the crosslinking time, the higher the degree of crosslinking, and the crosslinked collagen fibers. The weaker the mobility, the stronger the anti-degradation ability, the slower the degradation; the smaller the cross-linking agent concentration, the shorter the time, the weaker the cross-linking degree, the stronger the mobility of the cross-linked collagen fibers, and the anti-degradation ability.
  • concentration the better, such as glutaraldehyde, pent
  • concentration of dialdehyde as a crosslinking agent is determined in consideration of the nature of glutaraldehyde itself.
  • the effect of the method has an effect, so the beneficial effects of the invention are as follows: (1) effective inactivation of the collagen material, greatly reducing the risk of virus transmission, while retaining the mechanical properties of the collagen material after chemical crosslinking; 2) retaining the structure of the natural collagen helix region, retaining its biological activity, which is conducive to tissue adsorption and growth; (3)
  • the method is simple and effective.
  • Example 1 is a comparison chart of thermal shrinkage temperatures of Example 1 and Comparative Example 1 and Comparative Example 2;
  • Example 2 is a comparison diagram of tensile strength of Example 1 and Comparative Example 1 and Comparative Example 2;
  • Figure 3 is a comparison of heat shrinkage temperatures of Examples 2-4 and Comparative Examples 3-7;
  • Figure 4 is a graph comparing tensile strengths of Examples 2-4 and Comparative Examples 3-7.
  • Pretreatment Select the fresh bovine pericardium that has passed the inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • the bovine pericardium treated in the step 1) is immersed in a glutaraldehyde aqueous solution having a concentration of 0.5% to 1% at room temperature (25 ° C) for chemical crosslinking for not less than 1 h.
  • Pretreatment Select the fresh bovine pericardium that has passed the inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • the bovine pericardium treated in the step 1) is immersed in a glutaraldehyde aqueous solution having a concentration of 0.5% to 1% at room temperature (25 ° C) for chemical crosslinking for not less than 1 h.
  • Pretreatment Select the fresh bovine pericardium that has passed the inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 3 The fetal calf treated in step 2) was completely immersed in a 1 mol/L NaOH solution at room temperature (25 ° C) for 0.5-1 hour for inactivation treatment.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 3 The fetal calf treated in step 2) was completely immersed in a 1 mol/L NaOH solution at room temperature (25 ° C) for 0.5-1 hour for inactivation treatment.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 3 Inactivation: The calf treated in step 2) was inactivated by electron beam irradiation at room temperature (25 ° C) at a dose of 20 kGy.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 2) Inactivation: The fetal calf treated in step 1) was completely immersed in a 1 mol/L NaOH solution at room temperature (25 ° C) for 0.5-1 hour for inactivation treatment.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 2) Inactivation: The fetal calf treated in step 1) was completely immersed in a 1 mol/L NaOH solution at room temperature (25 ° C) for 0.5-1 hour for inactivation treatment.
  • Pretreatment Select fresh stevia that is qualified for inspection and quarantine, and remove only excess fat, blood and other impurities on the tissue by physical methods such as hand tearing, scissors and tweezers.
  • step 2) Inactivation: The calf treated in step 2) was inactivated by electron beam irradiation at room temperature (25 ° C) at a dose of 20 kGy.
  • the present invention performs the thermal shrinkage temperature and tensile strength tests on the bovine pericardium treated in Example 1 and Comparative Examples 1, 2, respectively, wherein Example 1 cross-links the bovine pericardium. - The order of inactivation was processed. Comparative Example 1 only cross-linked the bovine pericardium, and Comparative Example 2 processed the bovine pericard in the order of inactivation-crosslinking.
  • the present invention also performs the thermal shrinkage temperature and tensile strength tests on the treated fetal cows of Examples 2, 3 and Comparative Examples 3-6, respectively, wherein the crosslinking agent of Example 2 and Comparative Examples 3 and 4 is Denacol.
  • Example 2 cross-linking fetal calf - The order of inactivation was processed.
  • Comparative Example 3 only the calfskin was cross-linked, and the proportion of 4 was inactivated for the calfskin - The order of cross-linking was treated; the cross-linking agent of Example 3 and Comparative Examples 5, 6 was EDC, and Example 3 was cross-linked to the calfskin - The order of inactivation was treated.
  • Comparative Example 5 only cross-linked the calfskin, and Comparative Example 6 treated the calfskin in the order of inactivation-crosslinking.
  • Fig. 4 The tensile strength test results are shown in Fig. 4. It can be seen from the figure that only the inactivated collagen material of Example 2 has the best mechanical properties, and the mechanical properties of Example 3 are not greatly reduced, while Comparative Example 4 and Comparative Example The mechanical properties of 6 are greatly reduced. It can be seen that the treatment with sodium hydroxide will cause the peptide bond to be destroyed and hydrolyzed, thereby greatly reducing the mechanical properties of the material. In addition, although the mechanical properties of Comparative Example 5 are relatively high, it has not been inactivated. Treatment, with the risk of viral transmission.
  • the present invention also performs thermal shrinkage temperature and tensile strength tests on the treated fetal cows of Example 4 and Comparative Example 7, respectively, wherein Example 4 cross-links the fetal calfs. - The order of electron beam irradiation inactivation was processed, and Comparative Example 7 was processed in the order of electron beam irradiation inactivation-crosslinking.
  • the method of the present invention has the characteristics of effective inactivation while retaining mechanical properties and biological activity, and the method is simple and effective.

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Abstract

一种简单高效且成本低廉的灭活胶原材料的制备方法：先对天然胶原进行适当的化学交联后，再对其进行灭活处理。根据此方法获得了一种安全有效的胶原材料，作为医用胶原材料可供植入，供受损部位周围的细胞进入其中生长，促进受损组织愈合。该制备方法对胶原材料进行了有效灭活，极大地降低了病毒传播风险，同时保留了化学交联后的胶原材料的力学性能；保留了天然胶原螺旋区结构，保留其生物活性，有利于组织吸附和进入生长，方法简易有效。

Description

一种灭活胶原材料及其制备方法
技术领域
本发明涉及一种以天然胶原为原材料制成的灭活材料及其制备方法，特别涉及一种使用动物组织中的天然胶原制备而成的灭活材料及其制备方法。
背景技术
无论是器官移植还是组织缺损的修复，都存在同种异体器官或组织供体的严重不足，以及同种异体器官或组织移植而产生的排斥作用等负面影响。组织工程的兴起和迅猛发展给器官移植和软、硬组织的修复和重建带来了希望和福音。组织工程材料的研究与开发已经成为国际材料学研究的重要热点之一。
胶原蛋白或称胶原是由动物细胞合成的一种生物高分子，广泛存在于动物骨、软骨、肌腱、皮肤及其他结缔组织中，约占哺乳动物总蛋白的三分之一，是人体重要的细胞外基质成分。胶原应用于组织工程材料领域具有得天独厚的条件，其来源广泛成本低廉，具有良好的生物相容性、生物可降解性及低抗原性，亲水性好，其多孔构造有利于细胞的粘附，作为细胞外基质成分，对细胞具有诱导分化作用，可作为组织的支持物质，对细胞、组织乃至器官的正常功能以及创伤修复有重要影响，被制造成为各自促伤口愈合材料、支架材料等，医疗器械产品的制造领域有着不可替代的优势和相当广泛的应用。
虽然胶原材料有着诸多天然优势，但胶原大部分来源于动物，随着其在临床医疗产品里的运用日益广泛，病毒传播和免疫原性等方面的安全风险也日益增加。因此，来源组织的病毒、传染因子的携带风险以及对人体的潜在危害引起广泛关注。为确保最终医疗产品的安全性，需对动物源性原材料进行严格控制，在生产工艺中增加有效的病毒灭活或去除措施。
常用的病毒灭活方法有巴氏消毒法、干热灭活法、过氧乙酸 - 乙醇灭活法、乙醇灭活法、射线辐照灭活法、 NaOH 灭活法等。
巴氏消毒法是湿热灭活法之一，是国内外公认的病毒灭活方法，已在人血白蛋白制品中成功应用了数十年，灭活条件已很完善。该灭活方法可灭活脂包膜和部分非脂包膜病毒。同种异体植入性医疗器械在充分清洗血液及骨髓成分后，可运用该方法进行病毒灭活。采用该方法时应考虑温度分布的均一性和灭活时间。
干热灭活法主要用于冻干制品的病毒灭活。该方法的病毒灭活效果已为实验室验证和临床应用所肯定，可灭活 HIV 、乙型肝炎病毒（ HBV ）、丙型肝炎病毒（ HCV ）和甲型肝炎病毒（ HAV ）等多种病毒。干热灭活法的优势是可用于冻干后的同种异体植入性医疗器械。采用该方法时应考虑温度分布的均一性、冻干工艺和产品水分残留量等。
过氧乙酸具有极强的病毒灭活能力；乙醇可降低溶液的表面张力，有助于消毒剂完全渗透入同种异体植入性医疗器械中。过氧乙酸 - 乙醇灭活法是一种可靠的病毒灭活方法，可灭活脊髓灰质炎病毒（ PV ）、 HIV 、伪狂犬病病毒（ PRV ）；牛病毒性腹泻病毒（ BVDV ）；猪细小病毒（ PPV ）等多种病毒，其用于同种异体植入性医疗器械病毒灭活的效果已为实验室和临床试验所证实。采用该方法时应严格控制人用安全过氧乙酸残留量限制。
乙醇是临床上最为常用的表面消毒剂。该方法对多数有包膜病毒，如单纯疱疹病毒、 HIV 等具有灭活作用。在用于同种异体骨的病毒灭活时，应充分清洗血液及骨髓成分；同时因乙醇具有强挥发性，应采用浸泡处理，注意防止高温和明火。由于不同的方法对产品造成的影响不同，因此实际使用时需根据材料性质以及材料携带病毒种类进行选择。
射线辐照灭活法的主要优点包括灭活效率高、穿透力强、剂量易控制、无有害物质残留、无明显温度升高等。该方法主要通过破坏核酸而灭活病毒。由于病毒在不同介质中对射线的抗性不同，该方法用于同种异体植入性医疗器械的病毒灭活时，应尽量去除产品中的宿主组织和细胞，例如同种异体骨应充分清洗血液及骨髓成分。应根据产品的特性确定辐照剂量。采用该方法时应考虑辐照剂量的分布和灭活时间。
氢氧化钠一直被认为能有效去除蛋白和核酸。同时，它还能灭活大多数的病毒、细菌、酵母、真菌以及内毒素。使用氢氧化钠的一大好处在于有效、低成本、并能容易检测、清除和处理。氢氧化钠可有效灭活 BSE 病毒，这一病毒对大多数的处理手段都具有相当的抵抗，包括 360 ℃干烤一小时处理。同时由于氢氧化钠会对天然胶原中肽键造成破坏，使其水解，灭活的同时也会极大地降低胶原的力学性能。
除此以外，以上所述几种病毒灭活方法均会不同程度对胶原天然结构和功能造成破坏。
除去上述的病毒传播和免疫原性等方面的安全风险，胶原材料还因其容易降解，力学性能较差，在实际运用时往往需要对其进行适当改性从而提高其力学性能。目前胶原的改性方法主要有物理交联、化学交联及通过与其他高分子材料共混改性。物理方法通常有紫外线照射、重度脱水、γ射线照射和热交联等，优点是可避免外源性有毒化学物质进入胶原内，缺点是胶原交联程度低且难以获得均匀一致的交联。化学方法通常有使用化学试剂交联、侧链的修饰和生理活性物的固定化等，优点是比物理方法改性交联度高，且能获得均匀一致的交联，对调节、控制胶原的各种性质均匀良好的效果，缺点是引入外源有毒试剂，残留试剂难以清除。
目前最普遍的化学改性方法是化学试剂交联法。化学交联剂从功能上来分可以分为两类：第一类是具有双官能团的多官能团交联剂，例如：二异氰酸酯和环氧化合物 (BDDGE 、京尼平等 ) ，这类试剂在相邻的两条肽链间形成胺基桥键；第二类交联剂可以活化谷氨酸或天冬氨酸残基上的羧酸，使之可以与另一条肽链上的胺基反应形成酰胺结合，提供交联键。交联剂按交联键发生的部位可分为两种：一种是分子内交联，它是在同一个螺旋内两条肽链之间形成的交联键，这类交联键主要影响交联产物的变性温度和拉伸强度等性能；另一种是分子间交联，它是在两条相邻的螺旋间的肽链中形成的交联键。这类交联键主要影响交联产物的溶胀性和表面延伸性。当两条微纤维之间的距离小于交联剂分子的长度时，交联也可以在两条相邻的微纤维之间发生。
目前常用的化学交联剂有戊二醛、 1,6- 己二异氰酸酯 (HDI) 、碳化二亚胺 (EDC) 、叠氮二苯基磷 (DPPA) 、酰基叠氮化物、聚环氧化物以及京尼平等。
热皱缩温度、拉伸强度及抗降解强度是评价生物材料交联特性的重要指标，热皱缩温度和拉伸强度测试数值越高，表明力学性能越强。各种交联剂对生物材料的交联处理均会提高它们的热皱缩温度、拉伸强度及抗降解强度，而热皱缩温度、拉伸强度及抗降解强度的提高表明交联处理提高了制品的力学和生物稳定性。决定分子间交联反应程度的因素为蛋白质本身的构象以及所用交联剂的性质，不同性质的交联剂对胶原交联度的差别很大。
现有技术手段通常是先对提取的或者天然的胶原进行灭活，灭活过程往往会对胶原的化学、生物性能及结构造成不同程度的破坏，影响胶原的功能。因此，本发明寻求一种方法既对胶原进行有效灭活同时又能保留天然胶原三维结构，生物活性及力学性能，从而使得制备的胶原材料安全、有效。
发明内容
为解决上述灭活胶原材料制备过程中存在的问题，本发明的目的在于通过提供一种简单高效且成本低廉的灭活胶原材料的制备方法从而提供一种安全有效的胶原材料。更具体地说，本发明的主要目的是解决氢氧化钠灭活或/和电子束辐照灭活过程中对天然胶原的三维结构造成破坏导致其力学和生物性能降低的问题。
为实现上述目的，本发明采用的技术方案是：一种灭活胶原材料的制备方法：先对天然胶原进行适当的化学交联后，再对其进行氢氧化钠灭活或/和电子束辐照灭活处理；这样既能除去潜在的病毒威胁，进一步降低免疫原性，又能保留天然胶原的三维结构、生物特性和力学性能。通过上述方法制备的灭活胶原材料作为医用胶原材料可供植入，供受损部位周围的细胞进入其中生长，促进受损组织愈合。
进一步地，本发明所述灭活胶原材料的制备方法，包括以下具体步骤：
1）预处理：使用来自天然组织的胶原材料，仅用物理方法去除组织上的多余脂肪、血块等杂质；
2）化学交联：将步骤1）中处理后的胶原材料完全浸没在交联剂中对胶原进行固定；交联剂通过与胶原分子间较活泼的氨基及羧基发生化学反应，降低胶原基材的免疫原性，并提高基材本身的力学强度；
3）灭活：将步骤2）中处理后的胶原材料于室温（25℃）下完全浸没在NaOH溶液中进行灭活处理，或/和电子束辐照灭活处理；
4）清洗：将步骤3）中处理后的胶原材料清洗至中性或再进行化学交联一段时间后清洗至中性。
其中，步骤1）中所述物理方法包括如手撕、用剪刀剪、用镊子夹等；
优选地，步骤1）中所述胶原材料为猪、牛等动物的心包、皮肤、肌腱等含有较多胶原的组织。
其中，步骤2）中所述交联剂可以是醛类、碳亚二胺、环氧类、氰酸酯类等化合物，优选为戊二醛、乙二醇二缩水甘油醚（Denacol）或碳亚二胺盐酸盐（EDC）；当交联剂为戊二醛时，以戊二醛水溶液为佳，最佳浓度为0.5%-1%(w/v)。
进一步地，步骤2）中，当采用戊二醛为交联剂时，其交联时间为不少于1小时；当采用乙二醇二缩水甘油醚作为交联剂时，其交联时间为不少于1天或碳亚二胺盐酸盐作为交联剂时，其交联时间为不少于0.5h。
优选地，步骤3)中NaOH的浓度为1-2mol/L，浸泡时间为0.5-1小时。
优选地，步骤3)中电子束辐照灭活的辐照剂量为15-40kGy。
优选地，步骤4）中所述清洗至中性的具体操作为依次用缓冲液、生理盐水和注射用水漂洗至中性。
优选地，上述缓冲液为CBS（即碳酸钠 - 碳酸氢钠缓冲溶液）或/和PBS（磷酸缓冲盐溶液）。
通过控制交联剂浓度和交联时间可控制制得的胶原材料的最终性能，从理论上讲交联剂浓度越大，时间越长，则交联程度越高，经交联后的胶原纤维可移动性越弱，抗降解能力越强，降解越缓慢；交联剂浓度越小，时间越短，则交联程度越弱，经交联后的胶原纤维可移动性越强，抗降解能力越弱，降解越快；但是从实践中来说，要合理控制经济成本和时间成本，且对于某些交联剂来说并不是浓度越高越好的，如戊二醛，本发明中戊二醛作为交联剂的最佳浓度是考虑了戊二醛本身的性质确定的。
本发明人在对灭活胶原材料制备过程的研究中发现交联和灭活的顺序对最终产品的性能影响很大，此外交联剂和灭活剂的浓度、交联和灭活的时间也对方法的效果有影响，因此本发明的有益效果在于：（1）对胶原材料进行了有效灭活，极大降低了病毒传播风险，同时保留了化学交联后的胶原材料的力学性能；（2）保留了天然胶原螺旋区结构，保留其生物活性，有利于组织吸附和进入生长；（3）方法简易有效。
附图说明
图1是实施例1与对比例1、对比例2的热皱缩温度对比图；
图2是实施例1与对比例1、对比例2的拉伸强度对比图；
图3是实施例2-4与对比例3-7的热皱缩温度对比图；
图4是实施例2-4与对比例3-7的拉伸强度对比图。
具体实施方式
下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1：戊二醛/氢氧化钠
1）预处理：选用检验检疫合格的新鲜牛心包，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的牛心包于室温（25℃）下浸没在浓度为0.5%-1%的戊二醛水溶液中进行化学交联，时间不少于1h。
3）灭活：将步骤2）中处理后的牛心包于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
4）清洗：将步骤3）中处理后的胶原材料依次用PBS缓冲液，注射用生理盐水漂洗至中性。
对比例1：戊二醛
1）预处理：选用检验检疫合格的新鲜牛心包，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的牛心包于室温（25℃）下浸没在浓度为0.5%-1%的戊二醛水溶液中进行化学交联，时间不少于1h。
3）清洗：将步骤2）中处理后的胶原材料依次用PBS缓冲液，注射用生理盐水漂洗至中性。
对比例2：氢氧化钠/戊二醛
1）预处理：选用检验检疫合格的新鲜牛心包，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）灭活：将步骤1）中处理后的牛心包于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
3）化学交联：将步骤2）中处理后的牛心包于室温（25℃）下浸没在浓度为0.5%-1%的戊二醛水溶液中进行化学交联，时间不少于1h。
4）清洗：将步骤3）中处理后的胶原材料依次用PBS缓冲液，注射用生理盐水漂洗至中性。
实施例2：乙二醇二缩水甘油醚（Denacol）/氢氧化钠
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在乙二醇二缩水甘油醚中进行化学交联，时间不少于1天。
3）灭活：将步骤2）中处理后的胎牛皮于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
实施例3：碳亚二胺盐酸盐（EDC）/氢氧化钠
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在碳亚二胺盐酸盐中进行化学交联，时间不少于0.5h。
3）灭活：将步骤2）中处理后的胎牛皮于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
实施例4：乙二醇二缩水甘油醚（Denacol）/电子束辐照
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在乙二醇二缩水甘油醚中进行化学交联，时间不少于1天。
3）灭活：将步骤2）中处理后的胎牛皮于室温（25℃）下用电子束辐照进行灭活处理，剂量为20kGy。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
对比例3：Denacol
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在乙二醇二缩水甘油醚中进行化学交联，时间不少于1天。
3）清洗：将步骤2）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
对比例4：氢氧化钠/Denacol
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）灭活：将步骤1）中处理后的胎牛皮于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
3）化学交联：将步骤2）中处理后的胎牛皮于室温（25℃）下浸没在乙二醇二缩水甘油醚中进行化学交联，时间不少于1天。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
对比例5：EDC
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在碳亚二胺盐酸盐中进行化学交联，时间不少于0.5h。
3）清洗：将步骤2）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
对比例6：氢氧化钠/EDC
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）灭活：将步骤1）中处理后的胎牛皮于室温（25℃）下完全浸没在1mol/L的NaOH溶液中0.5-1小时进行灭活处理。
3）化学交联：将步骤2）中处理后的胎牛皮于室温（25℃）下浸没在碳亚二胺盐酸盐中进行化学交联，时间不少于0.5h。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
对比例7：电子束辐照/乙二醇二缩水甘油醚（Denacol）
1）预处理：选用检验检疫合格的新鲜胎牛皮，仅用物理方法如手撕，剪刀镊子等除去组织上的多余脂肪，血迹等杂质。
2）灭活：将步骤2）中处理后的胎牛皮于室温（25℃）下用电子束辐照进行灭活处理，剂量为20kGy。
3）化学交联：将步骤1）中处理后的胎牛皮于室温（25℃）下浸没在乙二醇二缩水甘油醚中进行化学交联，时间不少于1天。
4）清洗：将步骤3）中处理后的胶原材料依次用CBS缓冲液，注射用生理盐水漂洗至中性。
热收缩温度和拉伸强度测试
本发明对实施例1和对比例1、2处理好的牛心包分别进行热皱缩温度和拉伸强度测试，其中实施例1对牛心包按交联 - 灭活的顺序进行了处理，对比例1仅对牛心包进行了交联处理，对比例2对牛心包按灭活 - 交联的顺序进行了处理。
热皱缩测试结果如图1中所示，从图中可见三种处理均能使热皱缩温度升高，且实施例1和对比例1的热皱缩温度都超过了82℃。
拉伸强度测试结果如图2中所示，从图中可见只有实施例1中的灭活胶原材料其力学性能略微获得了提高，而两组对比例中的力学性能却大幅度下降。
综合上述测试的结果可以得出，先交联后灭活的处理对交联后的牛心包胶原材料的力学性能影响不大，不会产生严重的 肽键破坏、水解而极大降低胶原力学性能的问题 。
本发明也对实施例2、3和对比例3-6处理好的胎牛皮分别进行热皱缩温度和拉伸强度测试，其中实施例2和对比例3、4的交联剂为Denacol，实施例2对胎牛皮按交联 - 灭活的顺序进行了处理，对比例3仅对胎牛皮进行了交联处理，对比例4对胎牛皮按灭活 - 交联的顺序进行了处理；实施例3和对比例5、6的交联剂为EDC，实施例3对胎牛皮按交联 - 灭活的顺序进行了处理，对比例5仅对胎牛皮进行了交联处理，对比例6对胎牛皮按灭活 - 交联的顺序进行了处理。
热皱缩测试结果如图3中所示，从图中可见六种处理均能使热皱缩温度升高，而实施例2的热皱缩温度最高。
拉伸强度测试结果如图4中所示，从图中可见只有实施例2中的灭活胶原材料其力学性能最好，实施例3的力学性能也没有大幅下降，而对比例4和对比例6的力学性能却大幅度下降，可见先用氢氧化钠处理会造成肽键破坏和水解，从而大幅降低材料的力学性能；此外，对比例5的力学性能虽然比较高，但是其未经过灭活处理，具有病毒传播风险。
本发明还对实施例4和对比例7处理好的胎牛皮分别进行热皱缩温度和拉伸强度测试，其中实施例4对胎牛皮按交联 - 电子束辐照灭活的顺序进行了处理，对比例7按电子束辐照灭活-交联的顺序进行了处理。
热皱缩测试结果如图3中最后两列所示，从图中可见实施例4的热皱缩温度相对于新鲜胎牛皮获得了大幅提升，而对比例7的热皱缩温度变化不明显。
拉伸强度测试结果如图4中最后两列所示，从图中可见实施例4中的灭活胶原材料的力学性能相对于新鲜胎牛皮有所提高，而对比例7中的灭活胶原材料的力学性能远低于新鲜胎牛皮的力学性能，因此先用Denacol交联后再进行电子束辐照灭菌可很好地避免灭菌过程对肽键的破坏，从而维持了胎牛皮的良好力学性能。
综合上述测试的结果可以得出，先交联后灭活的处理对交联后的胎牛皮胶原材料的力学性能影响不大，不会产生严重的 肽键破坏、水解，而极大降低胶原力学性能的问题 。
因此，本发明的方法具有有效灭活、同时保留力学性能和生物活性、方法简易有效的特点。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (10)

  1. 一种灭活胶原材料的制备方法，其特征在于先对天然胶原进行适当的化学交联后，再对其进行灭活处理。
  2. 根据权利要求1所述的灭活胶原材料的制备方法，其特征在于包括以下具体步骤：
    1）预处理：使用来自天然组织的胶原材料，仅用物理方法去除组织上的杂质；
    2）化学交联：将步骤1）中处理后的胶原材料完全浸没在交联剂中对胶原进行固定；
    3）灭活：将步骤2）中处理后的胶原材料进行灭活处理；
    4）清洗：将步骤3）中处理后的胶原材料清洗至中性或再进行化学交联一段时间后清洗至中性。
  3. 根据权利要求2所述灭活胶原材料的制备方法，其特征在于步骤1）中所述物理方法为手撕、用剪刀剪或用镊子夹。
  4. 根据权利要求2所述灭活胶原材料的制备方法，其特征在于步骤1）中所述胶原材料为猪或牛的心包、皮肤或肌腱。
  5. 根据权利要求2所述灭活胶原材料的制备方法，其特征在于步骤2）中所述交联剂为醛类、碳亚二胺、环氧类或氰酸酯类。
  6. 根据权利要求2所述灭活胶原材料的制备方法，其特征在于步骤2）中所述灭活处理的方法为氢氧化钠灭活或/和电子束辐照灭活。
  7. 根据权利要求6所述的灭活胶原材料的制备方法，其特征在于所述氢氧化钠的浓度为1-2mol/L，浸泡时间为0.5-1小时。
  8. 根据权利要求6所述灭活胶原材料的制备方法，其特征在于所述电子束辐照灭活的辐照剂量为15-40kGy。
  9. 根据权利要求2所述灭活胶原材料的制备方法，其特征在于步骤4）中所述清洗至中性的具体操作为依次用缓冲液、生理盐水和注射用水漂洗至中性。
  10. 根据权利要求1-9所述灭活胶原材料的制备方法制备而得的灭活胶原材料。
PCT/CN2015/077898 2015-04-30 2015-04-30 一种灭活胶原材料及其制备方法 WO2016172894A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034910A1 (en) * 1995-05-04 1996-11-07 The University Of North Carolina At Chapel Hill Cross-linking of proteins, including collagen, using high osmolality storage medium
CN102533913A (zh) * 2011-12-23 2012-07-04 南昌大学 一种采用共固定化双酶水解鱼鳞胶原蛋白制备抗氧化活性肽的方法
CN103146789A (zh) * 2013-02-28 2013-06-12 珠海博康药业有限公司 一种胶原蛋白的制备方法
CN103333508A (zh) * 2013-06-28 2013-10-02 陕西巨子生物技术有限公司 一种注射用胶原蛋白水凝胶及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034910A1 (en) * 1995-05-04 1996-11-07 The University Of North Carolina At Chapel Hill Cross-linking of proteins, including collagen, using high osmolality storage medium
CN102533913A (zh) * 2011-12-23 2012-07-04 南昌大学 一种采用共固定化双酶水解鱼鳞胶原蛋白制备抗氧化活性肽的方法
CN103146789A (zh) * 2013-02-28 2013-06-12 珠海博康药业有限公司 一种胶原蛋白的制备方法
CN103333508A (zh) * 2013-06-28 2013-10-02 陕西巨子生物技术有限公司 一种注射用胶原蛋白水凝胶及其制备方法

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