WO2021143876A1 - 一种干燥胶原基生物材料的灭菌方法 - Google Patents

一种干燥胶原基生物材料的灭菌方法 Download PDF

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WO2021143876A1
WO2021143876A1 PCT/CN2021/072251 CN2021072251W WO2021143876A1 WO 2021143876 A1 WO2021143876 A1 WO 2021143876A1 CN 2021072251 W CN2021072251 W CN 2021072251W WO 2021143876 A1 WO2021143876 A1 WO 2021143876A1
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solution
collagen
sterilization
based biological
biological materials
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PCT/CN2021/072251
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French (fr)
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王云兵
郭高阳
金林赫
雷洋
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吉林启明皓月生物科技有限公司
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Publication of WO2021143876A1 publication Critical patent/WO2021143876A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof

Definitions

  • the invention belongs to the technical field of sterilization methods, and specifically relates to a sterilization method for dried collagen-based biological materials.
  • sterilization is a crucial process before implantation. If the sterilization is not complete, the residual bacteria will be carried into the human body by the implantable biological materials, which will cause fever, infection and inflammation. If biological materials come into contact with blood, bacteria will spread rapidly, causing widespread infections, and even life-threatening; at the same time, side reactions caused by bacteria will also cause damage to the performance of biological materials.
  • the sterilization of dried collagen-based implantable biomaterials is even more difficult. Due to the stability of its own structure, dried collagen-based biomaterials cannot be sterilized by conventional sterilization operations such as high temperature and high pressure, nor can they be sterilized by radioactive rays. Therefore, efficient and non-destructive sterilization of dry collagen-based implantable biomaterials is of great significance.
  • Biological heart valves will always be stored in glutaraldehyde aqueous solution before use to meet the sterilization requirements, but long-term soaking of glutaraldehyde will accelerate the calcification process of biological valves, affect their structural stability and performance, and reduce their service life. At the same time, the doctor needs to wash it repeatedly before use, which increases the preparation time and steps before the operation. Since there is no need to soak in the glutaraldehyde solution all the time, the sterilization process of the dried valve is very important.
  • dry valves are sterilized by ethylene oxide.
  • ethylene oxide is a toxic gas at room temperature, causing potential hazards to operators; the residue after sterilization of ethylene oxide is highly corrosive and may cause damage to the structure of the material.
  • ethylene oxide sterilization requires professional equipment support, which increases operational difficulty and production costs.
  • the present invention provides a sterilization method for dried collagen-based biomaterials, which is simple and quick to operate, does not require professional sterilization equipment, and effectively solves the problem that the sterilization process affects structural stability and performance, The problems of incomplete sterilization, difficult operation and low production cost.
  • the technical solution adopted by the present invention to solve its technical problems is to provide a sterilization method for dried collagen-based biological materials, which includes the following steps:
  • step (2) The secondary sterilized collagen-based biomaterial obtained in step (2) is immersed in an organic solvent at a temperature of 20-40°C, soaked for 0.5-48h, and then dehydrated and dried to obtain a dried collagen-based biomaterial.
  • step (1) is not necessary, but is an option of a specific implementation plan, which can be selected according to the implementation situation.
  • the antibiotic solution is one or more of vancomycin hydrochloride solution, gentamicin solution, penicillin solution, cefoxitin solution, streptomycin solution, chloramphenicol solution or amphotericin B solution, And the amount of antibiotic solution added is 0.2 ⁇ 2mL/mg.
  • the concentration of vancomycin hydrochloride solution is 20 ⁇ 60mg/L
  • the concentration of gentamicin solution is 6 ⁇ 15mg/L
  • the concentration of penicillin solution is 15 ⁇ 30mg/L
  • the concentration of cefoxitin solution is 200 ⁇ 300mg/L.
  • the concentration of streptomycin solution is 15-30mg/L
  • the concentration of chloramphenicol solution is 15-30mg/L
  • the concentration of amphotericin B solution is 20-40mg/L.
  • the pH of the peroxyacetic acid solution needs to be adjusted to 7.3 ⁇ 0.5 before being added, and then filtered through a 0.22 ⁇ m filter; and the addition amount of the peroxyacetic acid solution is 0.4 ⁇ 1mg/mg.
  • the organic solvent includes glycerin and alcohol solution, and the volume ratio of glycerin and alcohol solution is 1-50:20-100.
  • the alcohol solution is one or more of ethanol, isopropanol, methanol, 2-propanol or n-propanol.
  • step (1) a sterile cleaning solution is used for cleaning, a total of 2 cleanings, the first cleaning is 30 minutes, and the second cleaning is 12 hours.
  • step (2) a sterile cleaning solution is used for cleaning, a total of 3 cleanings, the first 2 cleanings are 30 minutes, and the third cleaning is 12 hours.
  • the antibiotic solution or the peroxyacetic acid solution can be dissolved in an organic solvent, and the dehydration and drying step can be performed.
  • the collagen-based biomaterial is animal tissue or an acellular matrix.
  • animal tissue includes one or more of pericardium, heart valve, intestinal membrane, meningeal membrane, lung membrane, blood vessel, small intestine, large intestine, skin or ligament.
  • Dry collagen-based biomaterials can be used to prepare thoracotomy biological valves, interventional biological valves, tissue engineered valves, artificial blood vessels, tissue engineered blood vessels, artificial skin, tissue engineered skin, artificial myocardial patch, artificial dura mater, artificial hernia patch, etc.
  • the present invention has the following advantages:
  • the sterilization method provided by the present invention is first sterilized by an antibiotic solution, then sterilized by peracetic acid for a second time, and finally immersed in an organic solvent, then dehydrated and dried.
  • Multiple antibiotic solutions can improve the sterilization effect while delaying Or to avoid the development of drug resistance, the peracetic acid treatment can further play the role of sterilization.
  • the sterile cleaning solution is thoroughly cleaned in a sterile environment; professional and expensive sterilization equipment is not required during the sterilization process, and the operation is simple and quick. It will not affect the structural stability and mechanical properties of the collagen-based biomaterials, and the sterilization is thorough, effectively reducing the inflammation and infection caused by bacteria, and at the same time ensuring the performance of the biomaterials.
  • the antibiotic mixed solution and peracetic acid solution can significantly reduce the endotoxin content and effectively sterilize the dry collagen-based implantable biological materials; non-toxic reagents are used in the sterilization process, which will not affect the dry collagen-based biomaterials.
  • the structural stability and use performance of the implanted biomaterials do not need to be equipped with professional sterilization equipment, reducing costs.
  • Figure 1 is a schematic diagram of residual endotoxin content in collagen-based biomaterials after different sterilization treatments
  • Figure 2 shows the degradation rate of collagenase in collagen-based biomaterials after different sterilization treatments.
  • a method for sterilizing dried collagen-based biological materials including the following steps:
  • step (3) Dehydration and drying: the collagen-based biological material obtained in step (2) after secondary sterilization is immersed in an organic solvent at a temperature of 20°C.
  • the organic solvent is a mixture of glycerin and ethanol in a 2:3 volume ratio. , Soaked for 5h, dehydrated and dried to obtain a dried collagen-based biomaterial.
  • a method for sterilizing dried collagen-based biological materials including the following steps:
  • step (3) Dehydration and drying: The collagen-based biological material obtained in step (2) after secondary sterilization is immersed in an organic solvent at a temperature of 30°C.
  • the organic solvent is a mixture of glycerin and ethanol in a 2:3 volume ratio. , Soaked for 24h, dehydrated and dried to obtain a dried collagen-based biomaterial.
  • a method for sterilizing dried collagen-based biological materials including the following steps:
  • step (3) Dehydration and drying: The collagen-based biomaterials obtained in step (2) after secondary sterilization are immersed in an organic solvent at a temperature of 30°C.
  • the organic solvent is glycerol, ethanol and isopropanol in a volume ratio of 2:2:1
  • the mixed liquid is soaked for 24 hours and then dehydrated and dried to obtain a dried collagen-based biological material.
  • a method for sterilizing dried collagen-based biological materials including the following steps:
  • step (3) Dehydration and drying: The collagen-based biological material obtained in step (2) after secondary sterilization is immersed in an organic solvent at a temperature of 40°C.
  • the organic solvent is a mixture of glycerol, ethanol and methanol in a volume ratio of 2:2:1. The resulting mixture is soaked for 48 hours and then dehydrated and dried to obtain a dried collagen-based biomaterial.
  • a method for sterilizing dried collagen-based biological materials including the following steps:
  • the mixed antibiotic solution is dissolved in an organic solvent.
  • the organic solvent includes the following components in volume percentage: glycerol 10%, ethanol 60%, isopropanol 30%, and then soak the collagen-based biological material in the antibiotic solution, 37 Incubate at °C for 24h, then wash with sterile water twice, the first wash is 30min, and the second wash is 12h;
  • step (2) immerse the collagen-based biomaterial obtained in step (1) in an organic solvent with a concentration of 0.05% peroxyacetic acid.
  • the organic solvent includes the following components in volume percentage: glycerol 10%, ethanol 60%, and isopropanol 30 %, shake for 3 hours at room temperature, then wash with sterile water 3 times, the first 2 washes for 30 minutes, and the third wash for 12 hours; finally, place it in a ventilated place and air-dry to obtain a dried collagen-based biomaterial.
  • tissue-engineered blood vessel into an antibiotic solution mixed with vancomycin hydrochloride, gentamicin, cefoxitin and amphotericin B in a volume ratio of 1:1:1:1, and soak at 37°C 24h, then wash with sterile water for 3 times, the first 2 washes for 30 minutes, and the third wash for 12 hours; then soak the collagen-based biological material in an organic solvent, the organic solvent includes the following components in volume percentage: glycerol 15%, ethanol 50%, 35% isopropanol, and finally air-dried in a ventilated place to obtain dried collagen-based biomaterials.
  • the tissue engineered blood vessel in 0.05% peracetic acid solution, shake for 3 hours at room temperature, then wash with sterile water 3 times, the first 2 washes for 30 minutes, and the third wash for 12 hours; then soak the collagen-based biomaterials in organic
  • the organic solvent includes the following components in volume percentage: 30% glycerin, 50% ethanol, 20% isopropanol) and finally placed in a ventilated place to air dry to obtain a dried collagen-based biomaterial.
  • Example 5 The residual endotoxin content and collagenase degradation rate in Example 5, Comparative Examples 1 to 2 and the untreated collagen-based biomaterials were determined. The results are shown in Figure 1 and Figure 2 respectively. Among them, Comparative Example 1 corresponds to Example 1. Ratio 2 corresponds to Example 2, and Example 5 corresponds to Example 3. It can be seen from Figure 1 that the sterilization effect is poor when using antibiotic solution alone; the effect is stronger when using peracetic acid solution alone; second, the combined sterilization effect of antibiotic solution and peracetic acid solution is the most significant, and sterilization The effect is obviously stronger than 75% ethanol immersion sterilization.
  • the combination of antibiotic solution and peracetic acid solution has the best sterilization effect, which can effectively kill bacteria without destroying the structural stability of the dried collagen-based biomaterial.

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Abstract

本发明提供了一种干燥胶原基生物材料的灭菌方法，包括以下步骤：将胶原基生物材料浸泡在抗生素溶液中，然后清洗，得初次灭菌后胶原基生物材料；将灭菌后的胶原基生物材料浸泡在过氧乙酸溶液中，然后室温下震荡，再清洗，得二次灭菌后胶原基生物材料；将二次灭菌后胶原基生物材料浸泡在有机溶剂中，浸泡后脱水干燥，得干燥胶原基生物材料。本发明操作简单快捷，无需专业的灭菌设备，有效解决了灭菌过程影响结构稳定性和使用性能、灭菌不彻底、操作难度大和生产成本低的问题。

Description

一种干燥胶原基生物材料的灭菌方法 技术领域
本发明属于灭菌方法技术领域，具体涉及一种干燥胶原基生物材料的灭菌方法。
 背景技术
对于外源性可植入生物材料，灭菌是植入前至关重要的过程。如果灭菌不彻底，残留细菌被可植入生物材料带入人体内部，会引起发热、感染及炎症等反应。如果生物材料接触血液，细菌会迅速传播，造成大范围感染，甚至危及生命；同时细菌引起的副反应也会导致生物材料性能上的损坏。而干燥胶原基可植入生物材料的灭菌更加困难，由于本身结构的稳定性问题，干燥胶原基生物材料无法进行高温高压等常规灭菌操作，也无法使用放射性射线进行灭菌。因此高效、无损的对干燥胶原基可植入生物材料进行灭菌具有十分重要的意义。
生物心脏瓣膜使用前会一直保存在戊二醛水溶液中以达到灭菌要求，但戊二醛的长期浸泡会加速生物瓣膜的钙化过程，影响其结构稳定性和使用性能，降低使用寿命。同时，使用前需要医生反复清洗，增加了术前的准备时间和步骤。由于无需一直浸泡在戊二醛溶液中，因此干燥瓣膜的灭菌过程至关重要。目前，干燥瓣膜通过环氧乙烷灭菌。但环氧乙烷在室温下是一种有毒气体，对于操作工人造成潜在的危害；环氧乙烷灭菌后的残留物具有较强的腐蚀性，可能对于材料的结构造成破坏。此外，环氧乙烷灭菌需要专业的设备支持，增加了操作难度和生产成本。
 技术问题
针对现有技术中存在的上述问题，本发明提供一种干燥胶原基生物材料的灭菌方法，操作简单快捷，无需专业的灭菌设备，有效解决了灭菌过程影响结构稳定性和使用性能、灭菌不彻底、操作难度大和生产成本低的问题。
 技术解决方案
为实现上述目的，本发明解决其技术问题所采用的技术方案是：提供一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）初次灭菌：将胶原基生物材料浸泡在抗生素溶液中，在20~40℃温度下孵化12~48h，然后清洗，得初次灭菌后胶原基生物材料；
（2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.02~0.5wt%的过氧乙酸溶液中，然后室温下以120rmp速率震荡2~4h，清洗，得二次灭菌后胶原基生物材料；
（3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在20~40℃温度下浸泡在有机溶剂中，浸泡0.5~48h后脱水干燥，得干燥胶原基生物材料。
进一步，步骤（1）不是必须的，是一个具体实施方案的选项，可根据实施情况选用。
进一步，抗生素溶液为万古霉素盐酸盐溶液、庆大霉素溶液、青霉素溶液、头孢西丁溶液、链霉素溶液、氯霉素溶液或两性霉素B溶液中的一种或多种，且抗生素溶液添加量为0.2~2mL/mg。
进一步，万古霉素盐酸盐溶液浓度为20~60mg/L、庆大霉素溶液浓度为6~15mg/L，青霉素溶液浓度为15~30mg/L，头孢西丁溶液浓度为200~300mg/L，链霉素溶液浓度为15~30mg/L，氯霉素溶液浓度为15~30mg/L，两性霉素B溶液浓度为20~40mg/L。
进一步，过氧乙酸溶液加入前需调节其pH至7.3±0.5，再经0.22μm过滤器过滤；且过氧乙酸溶液添加量为0.4~1mg/mg。
进一步，有机溶剂包括甘油和醇类溶液，甘油和醇类溶液的体积比为1~50:20~100。
进一步，醇类溶液为乙醇、异丙醇、甲醇、2-丙醇或正丙醇中的一种或多种。
进一步，步骤（1）中采用无菌清洗液清洗，共清洗2次，第1次清洗30min，第2次清洗12h。
进一步，步骤（2）中采用无菌清洗液清洗，共清洗3次，前2次清洗30min，第3次清洗12h。
进一步，在步骤（1）或步骤（2）清洗后，可将抗生素溶液或过氧乙酸溶液溶于有机溶剂中，进行脱水干燥步骤。
进一步，胶原基生物材料为动物组织或脱细胞基质。
进一步，动物组织包括心包膜、心脏瓣膜、肠膜、脑膜、肺膜、血管、小肠、大肠、皮肤或韧带中的一种或几种。
干燥胶原基生物材料可用于制备开胸生物瓣膜、介入生物瓣膜、组织工程瓣膜、人工血管、组织工程血管、人工皮肤、组织工程皮肤、人工心肌补片、人工硬脑膜、人工疝补片等。
 有益效果
综上所述，本发明具备以下优点：
1、本发明提供的灭菌方法首先通过抗生素溶液进行一次灭菌，在通过过氧乙酸二次灭菌，最后在有机溶剂中浸泡后脱水干燥，多个抗生素溶液可以提高灭菌效果，同时延缓或避免抗药性的产生，过氧乙酸处理可以进一步起到灭菌的作用，无菌清洗液在无菌环境中彻底清洗；在灭菌过程中不需要专业昂贵的灭菌设备，操作简单快捷，不会对胶原基生物材料的结构稳定性及力学性能产生影响，灭菌彻底，有效降低细菌引发的炎症和感染等问题，同时保证了生物材料的使用性能。
2、抗生素混合溶液和过氧乙酸溶液处理后可以显著降低内毒素含量，对干燥胶原基可植入生物材料进行有效灭菌；灭菌过程中均采用无毒试剂，不会影响干燥胶原基可植入生物材料的结构稳定性和使用性能，且不需要配备专业的灭菌设备，降低成本。
 附图说明
图1为不同灭菌处理后胶原基生物材料中残余内毒素含量示意图；
图2为不同灭菌处理后胶原基生物材料中胶原蛋白酶降解率。
 本发明的实施方式
实施例1  
一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）初次灭菌：将组织工程血管浸泡在抗生素溶液中，抗生素溶液为40mg/L的万古霉素盐酸盐溶液，抗生素溶液添加量为0.2mL/mg，在20℃温度下孵化12h，然后采用无菌水清洗，第1次清洗30min，第2次清洗12h，得初次灭菌后胶原基生物材料；
（2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.02wt%的过氧乙酸溶液中，过氧乙酸溶液加入前需调节pH至7.3±0.5，再经0.22μm过滤器过滤，过氧乙酸溶液添加量为0.4mg/mg，然后室温下以120rmp速率震荡2h，再采用无菌水清洗，前2次清洗30min，第3次清洗12h，得二次灭菌后胶原基生物材料；
（3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在20℃温度下浸泡在有机溶剂中，有机溶剂为甘油和乙醇按体积比2:3混合而成的混合液，浸泡5h后脱水干燥，得干燥胶原基生物材料。
实施例2
一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）初次灭菌：将组织工程血管浸泡在抗生素溶液中，抗生素溶液为40mg/L万古霉素盐酸盐溶液、10mg/L庆大霉素溶液和23mg/L青霉素溶液按体积比1:1:1混合而成的混合液，抗生素溶液添加量为1mL/mg，在30℃温度下孵化30h，然后采用无菌水清洗，第1次清洗30min，第2次清洗12h，得初次灭菌后胶原基生物材料；
（2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.3wt%的过氧乙酸溶液中，过氧乙酸溶液加入前需调节pH至7.3±0.5，再经0.22μm过滤器过滤，过氧乙酸溶液添加量为0.7mg/mg，然后室温下以120rmp速率震荡3h，再采用无菌水清洗，前2次清洗30min，第3次清洗12h，得二次灭菌后胶原基生物材料；
（3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在30℃温度下浸泡在有机溶剂中，有机溶剂为甘油和乙醇按体积比2:3混合而成的混合液，浸泡24h后脱水干燥，得干燥胶原基生物材料。
实施例3
一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）初次灭菌：将组织工程血管浸泡在抗生素溶液中，抗生素溶液为40mg/L万古霉素盐酸盐溶液、10mg/L庆大霉素溶液、23mg/L青霉素溶液、250mg/L头孢西丁溶液、23 mg/L链霉素溶液、23 mg/L氯霉素溶液和30 mg/L两性霉素B溶液按体积比1:1:1:1:1:1:1混合而成的混合液，抗生素溶液添加量为1mL/mg，在30℃温度下孵化30h，然后采用无菌水清洗，第1次清洗30min，第2次清洗12h，得初次灭菌后胶原基生物材料；
（2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.3wt%的过氧乙酸溶液中，过氧乙酸溶液加入前需调节pH至7.3±0.5，再经0.22μm过滤器过滤，过氧乙酸溶液添加量为0.7mg/mg，然后室温下以120rmp速率震荡3h，再采用无菌水清洗，前2次清洗30min，第3次清洗12h，得二次灭菌后胶原基生物材料；
（3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在30℃温度下浸泡在有机溶剂中，有机溶剂为甘油、乙醇和异丙醇按体积比2:2:1混合而成的混合液，浸泡24h后脱水干燥，得干燥胶原基生物材料。
实施例4
一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）初次灭菌：将组织工程血管浸泡在抗生素溶液中，抗生素溶液为10mg/L庆大霉素溶液、23mg/L青霉素溶液、250 mg/L头孢西丁溶液、23 mg/L链霉素溶液和23 mg/L氯霉素溶液按体积比1:1:1:1:1混合而成的混合液，抗生素溶液添加量为2mL/mg，在40℃温度下孵化48h，然后采用无菌水清洗，第1次清洗30min，第2次清洗12h，得初次灭菌后胶原基生物材料；
（2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.5wt%的过氧乙酸溶液中，过氧乙酸溶液加入前需调节pH至7.3±0.5，再经0.22μm过滤器过滤，过氧乙酸溶液添加量为1mg/mg，然后室温下以120rmp速率震荡4h，再采用无菌水清洗，前2次清洗30min，第3次清洗12h，得二次灭菌后胶原基生物材料；
（3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在40℃温度下浸泡在有机溶剂中，有机溶剂为甘油、乙醇和甲醇按体积比2:2:1混合而成的混合液，浸泡48h后脱水干燥，得干燥胶原基生物材料。
实施例5
一种干燥胶原基生物材料的灭菌方法，包括以下步骤：
（1）将40 mg/L万古霉素盐酸盐溶液、10mg/L庆大霉素溶液、250mg/L头孢西丁溶液和30mg/L两性霉素B溶液按体积比1:1:1:1混合而成的抗生素溶液溶于有机溶剂中，有机溶剂包括以下体积百分比的组分：甘油10%、乙醇60%、异丙醇30%，再将胶原基生物材料浸泡在抗生素溶液中，37℃温度下孵化24h，然后用无菌水清洗2次，第1次清洗30min，第2次清洗12h；
（2）再将步骤（1）所得胶原基生物材料浸泡在浓度为0.05%过氧乙酸的有机溶剂中，有机溶剂包括以下体积百分比的组分：甘油10%、乙醇60%、异丙醇30%，室温下震荡3h，然后用无菌水清洗3次，前2次清洗30min，第3次清洗12h；最后置于通风处风干，得干燥胶原基生物材料。
对比例1
将组织工程血管放入由万古霉素盐酸盐、庆大霉素、头孢西丁和两性霉素B按体积比1:1:1:1混合而成的抗生素溶液中，37℃温度下浸泡24h，然后用无菌水清洗3次，前2次清洗30min，第3次清洗12h；再将胶原基生物材料浸泡在有机溶剂中，有机溶剂包括以下体积百分比的组分：甘油15%、乙醇50%、异丙醇35%，最后置于通风处风干，得干燥胶原基生物材料。
对比例2
将组织工程血管浸泡在0.05%的过氧乙酸溶液中，室温下震荡3h，然后用无菌水清洗3次，前2次清洗30min，第3次清洗12h；再将胶原基生物材料浸泡在有机溶剂中，有机溶剂包括以下体积百分比的组分：甘油30%、乙醇50%、异丙醇20%）最后置于通风处风干，得干燥胶原基生物材料。
测定实施例5、对比例1~2以及未经处理的胶原基生物材料中残余内毒素含量和胶原蛋白酶降解率，其结果分别见图1和图2；其中，对比例1对应实例1，对比例2对应实例2，实施例5对应实例3。由图1可知，单独使用抗生素溶液进行灭菌时，效果较差；单独使用过氧乙酸溶液灭菌时效果较强；二将抗生素溶液和过氧乙酸溶液联合灭菌效果最为显著，且灭菌效果明显强于75%乙醇浸泡灭菌。由图2可知，经过灭菌处理后，干燥胶原基生物材料的胶原蛋白酶降解率变化不大，证明这3种灭菌方法不会对材料的结构稳定性造成破坏，即抗生素溶液和过氧乙酸溶液不会对材料的结构稳定性造成破坏。
综上所述，抗生素溶液配合过氧乙酸溶液联合灭菌效果最好，可以有效杀灭细菌，且不会破坏干燥胶原基生物材料的结构稳定性。
虽然结合附图对本发明的具体实施方式进行了详细地描述，但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内，本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。

Claims (10)

  1. 一种干燥胶原基生物材料的灭菌方法，其特征在于，包括以下步骤：
    （1）初次灭菌：将胶原基生物材料浸泡在抗生素溶液中，在20~40℃温度下孵化12~48h，然后清洗，得初次灭菌后胶原基生物材料；
    （2）二次灭菌：将步骤（1）初次灭菌后的胶原基生物材料浸泡在浓度为0.02~0.5wt%的过氧乙酸溶液中，然后室温下以120rmp速率震荡2~4h，清洗，得二次灭菌后胶原基生物材料；
    （3）脱水干燥：将步骤（2）所得二次灭菌后胶原基生物材料在20~40℃温度下浸泡在有机溶剂中，浸泡0.5~48h后脱水干燥，得干燥胶原基生物材料。
  2. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述抗生素溶液为万古霉素盐酸盐溶液、庆大霉素溶液、青霉素溶液、头孢西丁溶液、链霉素溶液、氯霉素溶液或两性霉素B溶液中的一种或多种，且所述抗生素溶液添加量为0.2~2mL/mg。
  3. 如权利要求2所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述万古霉素盐酸盐溶液浓度为20~60mg/L、庆大霉素溶液浓度为6~15mg/L，青霉素溶液浓度为15~30mg/L，头孢西丁溶液浓度为200~300mg/L，链霉素溶液浓度为15~30mg/L，氯霉素溶液浓度为15~30mg/L，两性霉素B溶液浓度为20~40mg/L。
  4. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述过氧乙酸溶液加入前需调节其pH至7.3±0.5，再经0.22μm过滤器过滤；且所述过氧乙酸溶液添加量为0.4~1mg/mg。
  5. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述有机溶剂包括甘油和醇类溶液，所述甘油和所述醇类溶液的体积比为1~50:20~100。
  6. 如权利要求5所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述醇类溶液为乙醇、异丙醇、甲醇、2-丙醇或正丙醇中的一种或多种。
  7. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，步骤（1）中采用无菌清洗液清洗，共清洗2次，第1次清洗30min，第2次清洗12h。
  8. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，步骤（2）中采用无菌清洗液清洗，共清洗3次，前2次清洗30min，第3次清洗12h。
  9. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，在步骤（1）或步骤（2）清洗后，可将抗生素溶液或过氧乙酸溶液溶于有机溶剂中，进行脱水干燥步骤。
  10. 如权利要求1所述的干燥胶原基生物材料的灭菌方法，其特征在于，所述胶原基生物材料为动物组织或脱细胞基质。
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