WO2018107487A1 - 一种角膜的辐照灭菌方法及其灭菌角膜 - Google Patents

一种角膜的辐照灭菌方法及其灭菌角膜 Download PDF

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WO2018107487A1
WO2018107487A1 PCT/CN2016/110465 CN2016110465W WO2018107487A1 WO 2018107487 A1 WO2018107487 A1 WO 2018107487A1 CN 2016110465 W CN2016110465 W CN 2016110465W WO 2018107487 A1 WO2018107487 A1 WO 2018107487A1
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cornea
irradiation
irradiating
sterilization
refrigerant
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PCT/CN2016/110465
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English (en)
French (fr)
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刘靖
李志寒
李洁
詹晓亮
董晓鸥
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厦门大开生物科技有限公司
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Priority to PCT/CN2016/110465 priority Critical patent/WO2018107487A1/zh
Priority to CN201680077804.9A priority patent/CN109069697B/zh
Publication of WO2018107487A1 publication Critical patent/WO2018107487A1/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
    • A61L27/38—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 containing added animal cells

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  • the present invention relates to a method of sterilizing a cornea, and more particularly to a method of sterilizing a cornea by irradiation.
  • the corneas to which the present invention relates include allogeneic and xenogeneic corneas.
  • the cornea as a medical material product for implantation into the human body (whether it is allogeneic or xenogeneic) has extremely strict sterilization requirements.
  • the artificial cornea with animal cornea as a source is expanding in clinical application, and it is expected to become a widely used medical device product in corneal transplantation in a short period of time.
  • decellularized artificial corneas as implantable medical device products should have higher sterilization requirements.
  • the sterilization method disclosed in the early patents on the preparation method of xenogeneic cornea and related literature is a human corneal-based drug sterilization method.
  • the sterilization method of transplanting allogeneic cornea in China is performed by using tobramycin solution to wash and disinfect.
  • This method can only achieve the antibacterial effect, but can not achieve the effect of completely killing all microorganisms such as bacteria and viruses. .
  • the irradiation sterilization technology introduces the sterilization treatment of the cornea.
  • Irradiation sterilization is an effective method to kill most microorganisms by using electromagnetic waves generated by ionizing radiation.
  • the principle is that ionizing radiation directly destroys biological DNA and macromolecular proteins, leading to microbial death. It is one of the commonly used methods for sterilization of medical devices because it does not raise the temperature of the sterilized subject and has strong penetrating power.
  • some medical device products with high sterilization requirements must be irradiated by irradiation with a dose of not less than 25kGy cobalt 60. Therefore, in the artificial corneal preparation method disclosed after 2010, the artificial cornea is basically sterilized by the irradiation method of cobalt 60.
  • the Chinese patent of CN1041189957A proposes that the irradiation dose range is 14-25 kGy, and other patent documents at the same time are irradiated by irradiation with a dose of 25 kGy.
  • the temperature of the irradiation chamber is usually higher than 30 °C at room temperature during normal temperature irradiation sterilization. For example, when the room temperature reaches 30 ° C in summer in the south, the temperature in the irradiation chamber can reach about 50 ° C. At this temperature, the collagen of the cornea has been denatured during irradiation, and the physical and chemical properties of the cornea, especially the transparency, vary greatly. Even in the case where the room temperature is low in the north, the temperature in the irradiation chamber can reach about 30 °C. This temperature also has a large effect on maintaining the transparency of the cornea.
  • the Chinese Patent No. CN1041189957A discloses a radiation sterilization technique, that is, "the whole eyeball sealed in a glycerin bottle is transferred to an insulated container of -20 ° C, irradiated with gamma rays, and the irradiation dose is 25 kGy".
  • the temperature inside the heat preservation container is substantially close to the irradiation room temperature.
  • the corneal collagen is denatured and the transparency is poor.
  • this method is not suitable for dry cornea because the dried cornea cannot be preserved in glycerol, so there is no protection of corneal collagen fibers by glycerol as described in the patent.
  • the inventors conducted experiments on the dried lamellar cornea, and irradiated the dried lamellar cornea (non-glycerol storage state) in a -20 ° C insulated container, and the corneal collagen was also significantly denatured, and the corneal transparency was poor. .
  • the object of the present invention is to provide a method for irradiating a cornea by irradiating the cornea with a strict sterilization standard to minimize the change of the physical and chemical properties of the cornea, thereby ensuring that the cornea is irradiated and sterilized. Transparency, toughness, and hydrophilicity are hardly altered to facilitate the clinical use of irradiated sterilized corneas.
  • the object of the present invention is to achieve a corneal irradiation sterilization method in which a gamma ray generated by a cobalt 60 irradiation source irradiates a cornea; and the cornea is placed in an insulated container in which a refrigerant is placed for irradiation. Sterilization; the cornea is maintained at a low temperature below 0 °C throughout the irradiation process by the refrigerant.
  • the refrigerant is ice.
  • the ice is placed in an insulated container in the form of ice cubes or ice packs, and the cornea is placed in the refrigerant.
  • the initial temperature of the ice is at least lower than -18 ° C to -25 ° C, and the insulated container is in a sealed state.
  • the refrigerant is dry ice.
  • the dry ice can be placed in a vented insulated container with the cornea placed therein.
  • the refrigerant is liquid nitrogen.
  • the liquid nitrogen is a steel cylinder as an insulated container.
  • the irradiation dose is not more than 35 kgy. In a preferred embodiment, the optimal irradiation dose is 15 to 25 kgy.
  • the cornea is maintained in the refrigerant throughout the corneal irradiation.
  • the cornea is sealed and packaged, and the corneal sealed package is entirely placed in a low-temperature refrigerant for irradiation.
  • the cornea is a dry cornea or a non-dried cornea.
  • the dried cornea has a water content of less than 20%.
  • the cornea is first placed in a low-temperature refrigerant, and irradiation is started after the cornea is rapidly lowered to a low-temperature ambient temperature.
  • the heat retention device constitutes an outer package at the time of irradiation.
  • the irradiation sterilization is performed after the corneal decellularization treatment.
  • the irradiation sterilization method of the cornea of the present invention performs terminal sterilization of the cornea.
  • the present invention also provides a sterilized cornea obtained by the above-described irradiation sterilization method of the cornea.
  • the cornea is a dry cornea; the dried cornea has a moisture content of less than 20% and a light transmission of no less than 75%.
  • the cornea is irradiated in a low-temperature refrigerant, ensuring that the cornea is always in a low temperature environment not higher than 0 ° C, effectively avoiding further damage to the collagen fiber structure of the cornea during irradiation sterilization.
  • the most valuable characteristic "transparency" of the cornea is effectively maintained.
  • Figure 1 is a photograph of a corneal electron microscope after sterilization in the present invention.
  • Figure 2 is a comparison of corneal transparency after sterilization of Example 2 of the present invention and sterilization method in the background art
  • Fig. 3 is a half-year effect picture of corneal transplantation obtained by the sterilization method of the present invention.
  • the gamma ray generated by the cobalt 60 irradiation source irradiates the cornea; the cornea is placed in an insulated container in which the refrigerant is placed for irradiation sterilization; The agent keeps the cornea at a low temperature below 0 °C throughout the irradiation.
  • the cornea is maintained in the low temperature refrigerant throughout the irradiation sterilization.
  • ice is specifically used as the low-temperature refrigerant.
  • the ice can be directly used in an ice pack or ice cube having an initial temperature of at least -18 to -25 ° C.
  • the ice described in this embodiment can be produced by any conventional method, for example, by direct freezing or by adding a cold storage agent for freezing.
  • the solid water "ice" is used as the refrigerant, and as the irradiation time of the gamma ray is extended, the temperature of the ice is increased accordingly, but the initial temperature of the ice used in the embodiment is low.
  • the initial temperature of the ice is selected based on the irradiation dose used, and the lower initial temperature should be selected when the irradiation dose is higher. Tests have shown that in this embodiment, the temperature of the ice in the whole process of irradiation, even if it is increased, can be kept at a low temperature of less than -5 ° C during the entire irradiation process, thereby ensuring the heat preservation. A low temperature state substantially the same as the temperature of the refrigerant is maintained in the container.
  • the temperature in the holding container is kept at a low temperature lower than 0 ° C, further destruction of the collagen fiber structure of the cornea during irradiation sterilization is effectively prevented, thereby effectively maintaining the transparency of the cornea.
  • Tests have shown that with the irradiation sterilization method of the present embodiment, the transparency, toughness and hydrophilic force of the cornea after sterilization are hardly changed, and the adverse effects of irradiation on the physical and chemical properties of the cornea under normal temperature conditions are effectively avoided. .
  • the irradiation dose is not more than 35 kgy. Specifically, in the test example, the irradiation dose is preferably in the range of 5 to 25 kgy.
  • the ice as the low-temperature refrigerant may be placed in the heat-retaining container in the form of ice or ice, and the cornea is placed in the refrigerant ice.
  • the insulated container can be sealed to avoid the influence of the temperature of the irradiation chamber on the temperature of the ice inside the container.
  • the insulated container simultaneously constitutes an outer package in the state of corneal irradiation.
  • the cornea can be first placed in the ice in the heat preservation container, and the irradiation is started after the cornea rapidly drops to the low temperature environment temperature in the moisturizing container.
  • the method of the first embodiment is applicable to a non-dried cornea, such as a cornea in a glycerol-preserved state, and is also suitable for drying a cornea.
  • This Example 1 is applied to terminal sterilization of a corneal product (for example, a dried cornea, or a non-dried cornea in a glycerin-preserved state).
  • a corneal product for example, a dried cornea, or a non-dried cornea in a glycerin-preserved state.
  • the cornea is monolithically sealed and packaged, and the corneal monolithic sealed package is entirely placed in low temperature refrigerant ice for irradiation. Keep the original sealed package after sterilization, It can maintain the sterilization effect of product terminal sterilization for a long time.
  • Another embodiment of the present embodiment 1 is an intermediate sterilization process in the corneal preparation process.
  • the irradiation of the fresh cornea may be a full eyeball or a prepared full-thickness cornea or lamellar cornea.
  • the method of the present invention as an intermediate sterilization treatment of the cornea may partially increase the preparation cost of the cornea, the irradiation sterilization treatment is advantageous for prolonging the fresh-keeping period of the fresh whole or non-dry cornea, and is beneficial for avoiding transportation and preservation of the corneal material. And waste of raw materials caused by cross-infection during preparation.
  • the irradiation sterilization treatment is carried out during the pretreatment, and if the sterilizing state is maintained during the subsequent preparation, the irradiation dose can be lowered at the time of terminal sterilization.
  • This method of segmental irradiation sterilization can also reduce the adverse effects of irradiation sterilization on the physical and chemical properties of the cornea.
  • the effect of the method of the present embodiment is illustrated by the following specific experimental example data, which includes only the corneal transmittance, and does not include the sterilization effect.
  • test results show that with the irradiation sterilization method of the invention, the physicochemical properties of the cornea after sterilization are small, and the transparency, toughness and hydrophilic force are not substantially changed, and the irradiation of the cornea at normal temperature is effectively avoided. Adverse effects of physical and chemical properties.
  • the gamma ray generated by the cobalt 60 irradiation source irradiates the cornea; the cornea is placed in an insulated container in which the refrigerant is placed for irradiation sterilization; The agent keeps the cornea at a low temperature below 0 °C throughout the irradiation.
  • the refrigerant described in the second embodiment uses dry ice.
  • the refrigerant dry ice is disposed in a gas permeable heat preservation container, and the cornea is placed in the dry In the ice.
  • the heat insulating container constitutes an outer package when the cornea is irradiated while maintaining the low temperature state of the refrigerant dry ice. Since the dry ice is composed of solid carbon dioxide and has a low initial temperature, the inside of the heat preservation container is not easy to heat up quickly, and it is easy to obtain and maintain a low ambient temperature, so that the dry ice is used as a corneal irradiation sterilization refrigerant. Preferred embodiment.
  • the irradiation dose is not more than 35 kgy. Specifically, in the present embodiment, the optimal dose for irradiation is 15 to 25 kgy.
  • the cornea can be first placed in dry ice, and the irradiation is started after the cornea rapidly drops to a low temperature ambient temperature.
  • the sterilization method of the second embodiment is particularly suitable for drying the cornea.
  • the initial temperature of the refrigerant dry ice in the second embodiment is -78 ° C, and the temperature is not higher than -40 ° C in the whole process of sterilization, and is suitable for the dry cornea with a low water content, which is favorable for maintaining the cornea well.
  • Collagen fiber structure before sterilization The test data of the present inventors showed that the sterilization of the present Example 2 effectively prevented the adverse effects of irradiation on the physical and chemical properties of the cornea under normal temperature conditions. The transparency, toughness and hydrophilicity of the cornea after sterilization are substantially unchanged from those before sterilization. It is a preferred embodiment of terminal corneal sterilization.
  • the cornea before the irradiation sterilization, the cornea is firstly sealed and packaged, and the corneal monolithic sealed package is placed in the gas permeable heat preservation container as a low temperature refrigerant. Irradiation in ice. After the sterilization, the original single-piece sealed package is kept, and the sterilization effect of the terminal sterilization can be maintained for a long time.
  • the cornea before the irradiation sterilization, the cornea is directly sealed and packaged in the integral sealing device, and then the integral sealed package is placed in the ventilated insulated container for low temperature refrigeration.
  • the terminal is irradiated in dry ice. After sterilization, the whole sealed package is opened under aseptic conditions, and then sealed in a single piece to form a product state of the single-piece sealed package.
  • Another embodiment of this embodiment 2 is to sterilize the terminal of the non-dried cornea. Since the terminal product of the non-dried cornea is usually stored in glycerin, the dehydration of the cornea is completed during the storage period by the dehydration of glycerol. In the present embodiment, the corneal product sealed in glycerin is placed in dry ice for irradiation sterilization. Keep the original sealed package after sterilization to maintain the sterilization effect of the product terminal.
  • the test of the present invention proves that for the irradiation sterilization of the non-dried cornea in the glycerin storage state, the transparency of the cornea after sterilization in the present embodiment is higher than the irradiation sterilization in the heat preservation container of -20 ° C, which is far higher. Irradiation sterilization at room temperature.
  • Figure 2 is a comparison of the transparency of the cornea of this embodiment with several other radiation sterilizations.
  • the first is the result of fresh corneal irradiation at room temperature (similar to the American eye bank TBI As a result, the transparency was significantly reduced; the second was the result of the dry corneal irradiation of the present invention, and the transparency was maintained; the third was the irradiation result of the cornea preserved in glycerin at -20 ° C in an insulated container.
  • the cornea has turned yellow, although the transparency is slightly higher than the first normal temperature irradiation, but it has turned yellow, indicating significant collagen degeneration.
  • the second embodiment has the advantages of simple operation, low cost, and suitability for industrialization.
  • the irradiation sterilization method of the present invention is suitable for sterilization treatment of allogeneic corneas and xenogeneic corneas.
  • FIG. 3 is a photograph of a post-clinical transplant of the embodiment 2, and the artificial cornea obtained by the irradiation sterilization method of the second embodiment is shown in FIG. 2 as compared with the existing documented clinical effects. ), higher transparency was restored within a week after transplantation.
  • the gamma ray generated by the cobalt 60 irradiation source irradiates the cornea; the cornea is placed in an insulated container in which the refrigerant is placed for irradiation sterilization; Agent The cornea is kept at a low temperature below 0 ° C throughout the irradiation.
  • the refrigerant described in this embodiment employs liquid nitrogen. The gamma ray penetrates the liquid nitrogen to sterilize the cornea, and the cornea and the heat preservation container are kept at a low temperature by the refrigerant liquid nitrogen.
  • the heat preservation container is a steel bottle, and the cornea is placed in liquid nitrogen of the steel bottle.
  • the cornea is first placed in an integrally sealed package, and then the integrally sealed package is placed in a steel cylinder. Since liquid nitrogen is composed of liquid nitrogen, it has a lower initial temperature than ice and dry ice, and it is easy to maintain a low temperature in a cylinder.
  • the irradiation dose is not more than 35 kgy. Specifically, in the third embodiment, the optimum dose for irradiation is 15 to 35 kgy.
  • the cornea can be first placed in a packaged sealed packaging container, and then the packaging container is placed in liquid nitrogen, and the cornea is rapidly lowered to a low temperature state before irradiation is started.
  • the sterilization method of this embodiment 3 is particularly suitable for drying the cornea.
  • the dried cornea has a water content of the cornea of less than 20%.
  • the refrigerant liquid nitrogen temperature in the third embodiment is lower than that of the two refrigerants, ice and dry ice. Due to the dry cornea with a lower moisture content, the collagen fiber structure is substantially fixed relative to the non-dried cornea with a higher water content, so the ultra-low temperature of liquid nitrogen is much less destructive to the collagen fiber structure in the matrix layer than the non-drying. cornea.
  • the test data of the present invention shows that the sterilization method of the third embodiment effectively avoids the adverse effects of irradiation on the physical and chemical properties of the cornea under normal temperature conditions.
  • the transparency, toughness and hydrophilicity of the cornea after sterilization are substantially unchanged from those before sterilization.
  • This embodiment 3 is a preferred embodiment of terminal sterilization of dried corneal products.
  • Another embodiment of the third embodiment that is, the sterilization treatment of the non-dried cornea, is relatively special, has a certain risk, and is not suitable for use in production.
  • the third embodiment can solve the technical problem to be solved by the invention, that is, the damage caused by the irradiation sterilizing on the physical and chemical properties of the cornea, especially the "transparency” can be effectively avoided. Thereby, the technical effect of maintaining the most important characteristic "transparency" of the cornea under the condition of strict sterilization is achieved, thereby achieving the object of the present invention.
  • the sterilized cornea of the present invention is obtained by the method as described above.
  • the cornea of the present invention is a dry cornea.
  • the water content is less than 20% and the light transmittance is not less than 70%.

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Abstract

一种角膜的辐照灭菌方法，采用钴60辐照源产生的γ射线对角膜进行辐照；先将角膜置于填充制冷剂的保温容器中；通过制冷剂保证角膜在辐照全过程中保持在低于0℃的低温状态下，在γ射线辐照灭菌后的角膜在透明度、韧性和亲水力几乎不发生改变，有利于延长角膜的保存时间。

Description

一种角膜的辐照灭菌方法及其灭菌角膜
所属领域
本发明涉及一种角膜的灭菌方法，具体地讲是一种利用辐照方法对角膜进行灭菌的方法。本发明涉及的角膜包括同种异体和异种异体角膜。
背景技术
基于中国角膜供体的严重缺乏现状，随着我国组织工程角膜技术的发展，异种异体角膜在临床应用的需求增加，异种异体角膜替代领域的研究更是取得了举世瞩目的成就。特别是以猪角膜为供体来源的脱细胞猪板层角膜的应用成果被国外科学界誉为中国当代五项重大创新之一。目前国内市场提供的以猪角膜为供体来源的脱细胞猪板层人工角膜产品，其中部分产品通过了中国相关部门市场准入审批进入临床使用，为我国由于角膜供体缺乏而造成的“角膜移植危机”提供了有效解决方案和光明的前景。
角膜作为一种用于植入人体的医用材料产品(无论是同种异体还是异种异体)都有极其严格的灭菌要求。特别是以动物角膜作为来源的人工角膜在临床应用范围的不断扩大，有望在较短的时间内成为角膜移植中广泛使用医疗器械产品。但作为植入类医疗器械产品的脱细胞人工角膜应当具有更高的灭菌要求。在早期关于异种异体角膜制备方法的专利及相关文献中所公开的灭菌方法是基于人角膜的药物灭菌方法。例如，目前我国同种异体角膜的移植手术前的灭菌方法使用妥布霉素溶液冲洗消毒，这种方法仅能达到抑菌的作用，而不能达到完全杀灭细菌、病毒等所有微生物的作用。随着各种新的灭菌的技术的成熟及广泛的应用，其中辐照灭菌技术引入角膜的灭菌处理。
辐照灭菌是利用电离辐射产生的电磁波杀死大多数微生物的一种有效方法，其原理在于电离辐射直接破坏生物的DNA和大分子蛋白质，导致微生物死亡。由于其不升高灭菌对象的温度且穿透力强，是医疗器械灭菌的常用方法之一。我国对于一些灭菌要求较高的医疗器械产品要求必须通过剂量不低于25kGy钴60的辐照灭菌。所以，2010年以后所公开的人工角膜制备方法中基本上都是采用钴60的辐照方法对人工角膜灭菌。其中第CN1041189957A中国专利中提出辐照剂量范围14～25kGy之外，其它同时间的专利文献均采用剂量25kGy的辐照灭菌。
但是通过大量的角膜辐照灭菌试验结果来看，辐照灭菌后，角膜的透明度、生物力学特性均有比较明显的下降。而美国国际组织库(Tissue Banks International,TBI)于2015年10月发表的文章《Physical and Biological Characterization of the Gamma-Irradiated Human Cornea》中也曾提到，使用的TBI角膜进行辐照灭菌试验，并通过对比辐照前后角膜的透明度、韧性和亲水力变化的试验数据分析后指出，常温条件下对人来源的角膜供体进行辐照灭菌，伽马射线可以使细胞外基质中胶原的分子键发生变化，辐照后角膜的理化性能发生改变，导致辐照后角膜的透明度、韧性、亲水力与正常生理状态下角膜相比，发生明显变化。因此该发表的论文中提出“辐照灭菌不利于角膜临床灭菌使用”结论。
当前国内外对角膜辐照灭菌的相关研究数据显示，在常温辐照灭菌的过程中，辐照室的温度在常温下通常高于30℃。例如，在南方夏季室温达到30℃时，辐照室内的温度大约可达到50℃。而在这种温度下，角膜的胶原蛋白在辐照过程中已经发生变性，角膜理化性能、特别是透明度变化较大。即使在北方室温较低的情况下，辐照室内的温度也可达到30℃左右。这个温度下也同样会对保持角膜的透明度产生较大的影响。
第CN1041189957A中国专利公开了一种辐照灭菌技术，即“将密封保存在甘油瓶内的全眼球转至-20℃的保温容器内，用伽马射线进行辐照，辐照剂量25kGy”。其提出的技术效果是利用“甘油具有的保护胶原纤维作用，使得胶原纤维避免高强度伽马射线的破坏，使之更加透明”。但是实际辐照实验证明，保温容器即使存在-20℃的初始温度，由于受辐照室较高环境温度的影响，会随辐照时间的延长，保温容器内的温度也会受辐照室高于30℃内的环境温度的影响逐渐升高，保温容器无法在辐照全过程中保持该初始的低温状态，通常情况下辐照3小时后，保温容器内温度基本上接近辐照室内温度。如图2所示，采用上述甘油状态下辐照灭菌后，角膜胶原蛋白变性明显，透明度较差。
另外，该方法不适用于干燥角膜，因为干燥角膜不能在甘油中保存,所以不存在如专利所述的甘油对角膜胶原纤维的保护作用。本发明人通过对干燥板层角膜进行了实验，将干燥板层角膜(非甘油保存状态)在-20℃的保温容器内进行辐照灭菌后，角膜胶原蛋白也明显变性，角膜透明度比较差。
因此，建立一种有效且对角膜影响最小的灭菌方式，将极大促进我国人工角膜的产业规模化发展，满足临床应用。
发明内容
本发明的目的在于提供一种角膜的辐照灭菌方法，在保证角膜严格的灭菌标准的前提下，将角膜的理化性能的变化降到最小程度，从而确保角膜在辐照灭菌后其透明度、韧性和亲水力几乎不发生改变，以利于辐照灭菌角膜的临床使用。
本发明的目的是这样实现的，一种角膜的辐照灭菌方法，钴60辐照源产生的伽马射线对角膜进行辐照；将角膜置于放置有制冷剂的保温容器中进行辐照灭菌；通过制冷剂使得角膜在辐照全过程中保持在低于0℃的低温状态下。
在本发明制冷剂的一个可选例子中，所述的制冷剂是冰。在该例子中，所述的冰采用冰块或冰袋形态设置于保温容器，将角膜置于制冷剂中。所述冰的初始温度至少低于-18℃～-25℃，所述该保温容器呈密封状态。
在制冷剂的另一个可选例子中，所述的制冷剂是干冰。在该例子中，所述的干冰可设置于透气的保温容器中，将角膜置于其中。
在制冷剂的再一个可选例子中，所述的制冷剂是液氮。在该例子中，所述的液氮以钢瓶作为保温容器。
在本发明的角膜的辐照灭菌方法中，所述辐照剂量不大于35kgy。在一个优先例子中，所述辐照最佳剂量为15～25kgy。
在本发明的角膜的辐照灭菌方法中，对角膜辐照的全过程中角膜均保持在所述制冷剂中。
在本发明的角膜的辐照灭菌方法中，在进行辐照灭菌前，先将角膜进行密封包装，将角膜密封包装整体放置于低温制冷剂中进行辐照。
在本发明的角膜的辐照灭菌方法中，所述角膜为干燥角膜或非干燥角膜。
在本发明的角膜的辐照灭菌方法中，所述干燥角膜的含水率低于20％。
在本发明的角膜的辐照灭菌方法中，将角膜先置于低温状态制冷剂中，待角膜迅速降至低温环境温度后再开始进行辐照。
在本发明的角膜的辐照灭菌方法中，所述保温装置构成辐照时的外包装。
在本发明的角膜的辐照灭菌方法中，所述辐照灭菌在角膜脱细胞处理之后进行。
在本发明的角膜的辐照灭菌方法中，所述辐照灭菌方法对角膜的进行终端灭菌。
本发明还提供了一种通过上述角膜的辐照灭菌方法获得的灭菌角膜。在一个可选例子中，该角膜是干燥角膜；所述干燥角膜的含水率低于20％，透光率不低于75％。
众所周知，角膜的异体移植本身即存在病毒感染的潜在危险。因此标准化异体角膜，特别是异种异体角膜必须最大限度地降低潜在的感染机率。由于在生物材料中，γ射线照射是一种被广泛应用于生物材料中实现终端灭菌的有效方法。因此，在本发明中进行角膜的灭菌处理方法仍建立在辐照的基础上，可以确保取得非常好的灭菌效果，有效地降低了细菌、病毒感染的潜在危险，确保达到国家对角膜，特别是异种异体替代角膜的严格的灭菌和病毒灭活标准。
在本发明中，角膜在低温制冷剂中进行辐照，保证了角膜始终处于不高于0℃的低温环境中，有效地避免了在辐照灭菌过程中对角膜的胶原纤维结构的进一步破坏，从而有效保持了角膜的最宝贵的特性“透明度”。虽然目前尚无其它的研究结论表明，在制冷剂的全程低温条件下对角膜进行辐照所产生的影响。但是，本申请人大量的试验结果证明，采用本发明的辐照的灭菌方法，灭菌后的角膜的理化性能变化小，透明度、韧性和亲水力几乎不发生改变，有效地避免了常温状态下辐照对角膜理化性能产生的不利影响。
附图说明
下面结合附图对本发明及其具体实施方式及其效果作简单地介绍，下面的附图仅仅是选择本发明的一些具体试验例说明，而非本发明的全部。
图1本发明灭菌后角膜电镜下照片。
图2本发明实施例2与背景技术中灭菌方法灭菌后角膜透明度的比较；
图3本发明的灭菌方法获得的角膜移植手术后半年效果图片。
具体实施方式
下面将结合本发明实施例，对本发明技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本发明实施例1的角膜的辐照灭菌方法，钴60辐照源产生的伽马射线对角膜进行辐照；将角膜置于放置有制冷剂的保温容器中进行辐照灭菌；通过制冷剂使得角膜在辐照全过程中保持在低于0℃的低温状态下。
在本实施例1中较佳地选择是角膜在辐照灭菌的全过程中均保持在低温制冷剂中。
在本实施例1中，具体采用冰作为低温制冷剂。该冰可以直接采用冷冻室内制成，初始温度至少低于-18～-25℃的的冰袋或者冰块。本实施例中所述的冰可以由任意一种常规则的方法制作，例如可以进行直接冷冻、或者加入蓄冷剂进行冷冻。本实施例1中以固态的水“冰”作为制冷剂，随着伽马射线的辐照时间延长，冰的温度随之而有所上升，但由于本实施例中采用的冰的初始温度低于-18～-25℃，冰的初始温度的依据采用的辐照剂量进行选择，辐照剂量较高时应当选择较低的初始温度。试验证明，在本实施例中，在辐照的全程中冰的温度，即使有所升高，但在全部辐照过程中的基本上可以保持在低于-5℃的低温状态，从而确保保温容器内保持有与制冷剂温度基本相同的低温状态。由于保持保温容器内的温度处于低于0℃的低温状态，有效地避免了在辐照灭菌过程中对角膜的胶原纤维结构的进一步破坏，从而有效保持了角膜的透明度。试验证明，采用本实施例的辐照的灭菌方法，灭菌后的角膜的透明度、韧性和亲水力几乎不发生改变，有效地避免了常温状态下辐照对角膜理化性能产生的不利影响。
在本实施例1中，辐照剂量不大于35kgy。具体在本试验例中，辐照剂量较佳范围5～25kgy。
在本实施例1中，作为低温制冷剂的冰，可以采用冰块或冰袋形态设置于保温容器内，再将角膜置于制冷剂冰中。保温容器可呈密闭状态，避免辐照室温度升高对保容器内冰的温度的影响。该保温容器同时构成角膜辐照状态下的外包装。
在本实施例1中，可先将角膜先行置于保温容器内冰中，待角膜迅速降至保湿容器内的低温环境温度后再开始进行辐照。
本实施例1所述方法即适用于非干燥角膜，如甘油保存状态下的角膜，也适用于干燥角膜。本实施例1应用于角膜产品(例如干燥角膜，或者甘油保存状态下的非干燥角膜)的终端灭菌。在进行辐照灭菌前，先对角膜进行单片密封包装，将角膜单片密封包装整体置于低温制冷剂冰中进行辐照。灭菌后保持原密封包装， 可以长期维持产品终端灭菌的灭菌效果。
而本实施例1另一种实施方式是在角膜制备过程的中间灭菌处理。例如在对新鲜角膜的预处理过程中进行辐照灭菌处理，新鲜角膜可以是全眼球或者经过制备后的全层角膜或板层角膜。尽管本发明的方法作为角膜的中间灭菌处理会部分增加角膜的制备成本，但是通过辐照灭菌处理有利于延长新鲜全眼球或非干燥角膜的保鲜期，有利于避免角膜材料在运输、保存以及制备过程中的交叉感染造成的原材料浪费。
另外，在预处理过程中进行辐照灭菌处理，如果在其后的制备过程中保持较好的无菌状态，可以在终端灭菌时降低辐照剂量。这种分段辐照灭菌的方法也可以减小辐照灭菌对角膜理化性能产生的不利影响。
本实施例的方法效果通过如下具体实验例数据进行说明，本数据仅包括角膜透光率，不包括灭菌效果。
干燥角膜的实验例
辐照剂量(kgy) 5±2.0 15±3.0 20±4.0 25±5.0
角膜透光率(％) 78±1％ 78±1％ ≥75％ ≥70％
非干燥角膜的实验例
辐照剂量(kgy) 5±2.0 15±3.0 20±4.0 25±5.0
角膜透光率(％) 75±2％ 72±2％ 70±2％ 70±2％
试验结果表明，采用本发明的辐照的灭菌方法，灭菌后的角膜的理化性能变化小，透明度、韧性和亲水力基本上不发生改变，有效地避免了常温状态下辐照对角膜理化性能产生的不利影响。
实施例2
本发明实施例2的角膜的辐照灭菌方法，钴60辐照源产生的伽马射线对角膜进行辐照；将角膜置于放置有制冷剂的保温容器中进行辐照灭菌；通过制冷剂使得角膜在辐照全过程中保持在低于0℃的低温状态下。本实施例2中所述的制冷剂采用干冰。
本实施例2中，所述的制冷剂干冰设置于透气的保温容器中，将角膜置于干 冰中。该保温容器在保持制冷剂干冰的低温状态的同时构成角膜辐照时的外包装。由于干冰由固态的二氧化碳构成，具有较低的初始温度，保温容器内不易很快地升温，较容易获得并保持较低的环境温度，因此以干冰作为角膜辐照灭菌的制冷剂是本发明的较佳实施方式。
在本实施例2中，辐照剂量不大于35kgy。具体在本实施例中，辐照最佳剂量15～25kgy。
在本实施例2中，可先将角膜先置于干冰中，待角膜迅速降至低温环境温度后再开始进行辐照。
本实施例2所述灭菌方法特别适用于干燥角膜。本实施例2中的制冷剂干冰的初始温度在-78℃，在灭菌的全过程中，温度不高于-40℃，适用于含水率较低的干燥角膜，有利于很好地保持角膜灭菌前的胶原纤维结构。而本发明人的试验数据表明，采用本实施例2的灭菌地有效地避免了常温状态下辐照对角膜理化性能产生的不利影响。灭菌后的角膜的透明度、韧性和亲水力与灭菌前相比较基本上不发生改变。是角膜产品终端灭菌的较佳实施方式。
本实施例2的一种较佳的实施方式中，在进行辐照灭菌前，先对角膜进行单片密封包装，将角膜单片密封包装整体置于所述透气的保温容器中低温制冷剂冰中进行辐照。灭菌后保持原单片密封包装，可以长期维持产品终端灭菌的灭菌效果。
本实施例2的另一种可选择的实施方式中，在进行辐照灭菌前，将角膜直接密封包装于整体密封装置内，再将整体密封包装置于所述透气的保温容器中低温制冷剂干冰中进行终端辐照。灭菌后在无菌状态下打开整体密封包装后再进行单片密封包装，形成单片密封包装的产品状态。
本实施例2的另一种实施方式是对非干燥角膜的终端灭菌。由于非干燥角膜的终端产品通常保存在甘油中，利用甘油的脱水作用在保存期内完成角膜的脱水过程。而本实施方式中，将密封包装于甘油中的角膜产品置于干冰中进行辐照灭菌。灭菌后保持原密封包装，可以保持产品终端灭菌的效果。
本发明的试验证明，对于甘油保存状态下的非干燥角膜的辐照灭菌，本实施方式灭菌后角膜的透明度高于-20℃的保温容器中的辐照灭菌，更是远远高于常温下的辐照灭菌。图2是本实施例与其它几种辐照灭菌后角膜的透明度的对比。图2中从左至右：第一个为新鲜角膜常温辐照后结果(类似于美国眼库TBI的 结果)，其透明度明显下降；第二个为本发明干燥角膜辐照后结果，保持有很好的透明度；第三个为在甘油中保存的角膜在-20℃的保温容器中的辐照结果，角膜已经变黄，虽透明度略高于第一个常温辐照的情况，但是已经发黄，显示发生了明显的胶原蛋白变性。
本实施例2所示方法效果通过如下具体实验数据进行说明，本数据仅包括角膜透光率。
干燥角膜的实验例
辐照剂量(kgy) 5±2.0 15±3.0 20±4.0 25±5.0
角膜透光率(％) 80±2％ 80±2％ ≥75％ ≥75％
非干燥角膜的实验例
辐照剂量(kgy) 5±2.0 15±3.0 20±4.0 25±5.0
角膜透光率(％) 75±2％ 75±2％ 70±2％ 70±2％
大量的试验证明，本实施例2的灭菌后对角膜透明度所产生的影响明显小于现有技术中的辐照灭菌方法对角膜透明度的影响。虽然目前尚不能给出该效果所产生的原理，但是试验证明将角膜置于制冷剂干冰中辐照与仅在保温容器中辐照，辐照后角膜的透明度差异相当大。这也正是本发明所要解决辐照灭菌对角膜理化性能、特别是透明度产生的不利影响的问题，在保证临床灭菌的前提下，保持角膜最重要的特性“透明度”，从而达到本发明的目的。另外本实施例2还具有操作简单、成本低，适于产业化等优势。
本发明的上述技术效果显示，本发明的辐照的灭菌方法，适用于同种异体角膜和异种异体角膜的灭菌处理。
图3为本实施例2的一个临床移植术后照片，相对于现有的有文献记载的临床效果而言，采用本实施例2的辐照灭菌方法所获得的人工角膜(图2所示)，在移植术后在一周内即已经恢复了较高的透明度。
实施例3
本发明实施例3的角膜的辐照灭菌方法，钴60辐照源产生的伽马射线对角膜进行辐照；将角膜置于放置有制冷剂的保温容器中进行辐照灭菌；通过制冷剂 使得角膜在辐照全过程中保持在低于0℃的低温状态下。本实施例中所述的制冷剂采用液氮。伽马射线穿透液氮对角膜进行辐照灭菌，通过制冷剂液氮使得角膜及保温容器同时保持在低温状态下。
本实施例3中，所述的保温容器是钢瓶，将角膜置于钢瓶的液氮中。具体在本实施例3中，为操作方便，先将角膜置于一个整体密封的包装中，然后再将整体密封的包装置于钢瓶内。由于液氮由液态的氮构成，相对于含有冰和干冰而言，其有更低的初始温度，在钢瓶内很容易保持低温状态。
在本实施例3中，辐照剂量不大于35kgy。具体在本实施例3中辐照最佳剂量15～35kgy。
在本实施例中，可先将角膜装入包装密封的包装容器后，再将包装容器放入液氮中，角膜迅速降至低温状态后再开始进行辐照。
本实施例3所述灭菌方法特别适用于干燥角膜。具体在本实施例3中，所述干燥角膜为角膜的含水率低于20％。相较于冰和干冰两种制冷剂而言，本实施例3中的制冷剂液氮的温度更低。由于含水率较低的干燥角膜，相对于含水率较高的非干燥角膜其胶原纤维结构基本固定，因此液氮的超低温度对基质层内的胶原纤维结构产生的破坏性也远远小于非干燥角膜。
而本发明的试验数据表明，采用本实施例3的灭菌方法有效地避免了常温状态下辐照对角膜理化性能产生的不利影响。灭菌后的角膜的透明度、韧性和亲水力与灭菌前相比基本上不发生改变。本实施例3是干燥角膜产品终端灭菌的一个较佳实施方式。
本实施例3的另一种实施方式，即对非干燥角膜的灭菌处理，相对特殊，具有一定的危险性，不适于在生产使用。
本实施例所示方法效果通过如下具体实验数据进行说明如下：
干燥角膜的实验例
辐照剂量(kgy) 15±2.0 20±3.0 30±3.0 34±1.0
角膜透光率(％) ≥75％ ≥75％ 74±1％ 74±1％
大量的试验证明，本实施例3的提出的灭菌方法对于干燥角膜灭菌效果是比较好。在本实施3中，适于采用的相对于本发明实施例1和2中更高的辐照剂量范围，以达到更高的灭菌需求。
本实施例3同样可以解决本发明所要解决的技术问题即可以有效地避免辐照灭菌对角膜理化性能，特别是“透明度”所产生的所产生的破坏作用。从而在实现在满足严格灭菌的条件下，保持角膜最重要的特性“透明度”的技术效果，从而达到本发明的目的。
本发明所述灭菌角膜是通过如上所述的方法获得。特别本发明所述角膜是干燥角膜。其含水率低于20％，透光率不低于70％。
针对上述各实施方式的详细解释，其目的仅在于对本发明进行解释，以便于能够更好地理解本发明，但是，这些描述不能以任何理由解释成是对本发明的限制，特别是，在不同的实施方式中描述的各个特征也可以相互任意组合，从而组成其他实施方式，除了有明确相反的描述，这些特征应被理解为能够应用于任何一个实施方式中，而并不仅局限于所描述的实施方式。
[根据细则26改正07.03.2017]　

Claims (22)

  1. [根据细则26改正07.03.2017]　
    一种角膜的辐照灭菌方法，钴60辐照源产生的伽马射线对角膜进行辐照；其特征在于，将角膜置于放置有制冷剂的保温容器中进行辐照灭菌；通过制冷剂使得角膜在辐照全过程中保持在低于0°C的低温状态下。
  2. [根据细则26改正07.03.2017]　
    如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述的制冷剂是冰。
  3. [根据细则26改正07.03.2017]　
    如权利要求2所述角膜的辐照灭菌方法，其特征在于，所述的冰采用冰块或冰袋形态设置于保温装置中，将角膜置于其中。
  4. [根据细则26改正07.03.2017]　
    如权利要求2或3所述角膜的辐照灭菌方法，其特征在于，所述冰的初始温度至少低于-18°C〜-25°C;
  5. [根据细则26改正07.03.2017]　
    如权利要求2所述角膜的辐照灭菌方法，其特征在于，所述保温容器呈密封状态。
  6. [根据细则26改正07.03.2017]　
    如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述的制冷剂是干冰。
  7. [根据细则26改正07.03.2017]　
    如权利要求6所述角膜的辐照灭菌方法，其特征在于，所述的干冰设置于透气的保温装置中，将角膜置于其中。
  8. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述的制冷剂是液氮。
  9. 如权利要求8所述角膜的辐照灭菌方法，其特征在于，所述的制冷剂液氮以钢瓶作为保温容器。
  10. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述辐照剂量不大于35kgy。
  11. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述辐照最佳剂量15～25kgy。
  12. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，对角膜辐照的全过程中角膜均保持在所述制冷剂中。
  13. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述角膜包括同种异体或异种异体角膜。
  14. 如上述任意一所述角膜的辐照灭菌方法，其特征在于，在进行辐照灭菌前，先将角膜进行密封包装，将角膜密封包装整体置于低温制冷剂中进行辐照。
  15. 如上述任意一权利要求所述角膜的辐照灭菌方法，其特征在于，所述角膜为干燥角膜或非干燥角膜。
  16. 如权利要求14所述角膜的辐照灭菌方法，其特征在于，所述干燥角膜的含水率低于20％。
  17. 如上述任意一所述角膜的辐照灭菌方法，其特征在于，将角膜先置于低温状态制冷剂中，待角膜迅速降至低温环境温度后再开始进行辐照。
  18. 如权利要求1所述角膜的辐照灭菌方法，其特征在于，所述保温容器构成辐照时的外包装。
  19. 如上述任意一所述角膜的辐照灭菌方法，其特征在于，所述辐照灭菌在角膜脱细胞处理之后进行。
  20. 如权利要求1～18任意一所述角膜的辐照灭菌方法，其特征在于，所述辐照灭菌方法对角膜的进行终端灭菌。
  21. 一种灭菌角膜，其特征在于，所述该灭菌角膜是通过上述任一权利要求所述的方法获得。
  22. 如权利要求21所述灭菌角膜，其特征在于，所述角膜是干燥角膜。
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