WO2018058541A1 - 冷冻处理中保护生物组织的防冻剂及其制备方法 - Google Patents

冷冻处理中保护生物组织的防冻剂及其制备方法 Download PDF

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WO2018058541A1
WO2018058541A1 PCT/CN2016/101112 CN2016101112W WO2018058541A1 WO 2018058541 A1 WO2018058541 A1 WO 2018058541A1 CN 2016101112 W CN2016101112 W CN 2016101112W WO 2018058541 A1 WO2018058541 A1 WO 2018058541A1
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biological tissue
freezing
antifreeze
weight ratio
sodium polyacrylate
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PCT/CN2016/101112
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English (en)
French (fr)
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吴博贵
陈威明
陈正丰
王致又
吴文海
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台北荣民总医院
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Application filed by 台北荣民总医院 filed Critical 台北荣民总医院
Priority to US16/338,329 priority Critical patent/US11160271B2/en
Priority to EP16917291.3A priority patent/EP3520746A4/en
Priority to PCT/CN2016/101112 priority patent/WO2018058541A1/zh
Publication of WO2018058541A1 publication Critical patent/WO2018058541A1/zh
Priority to US17/397,506 priority patent/US11596145B2/en

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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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A61F7/02—Compresses or poultices for effecting heating or cooling

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  • the invention relates to an antifreeze and a preparation method thereof, in particular to a tissue for protecting a tissue during freezing treatment of human or animal tissues.
  • Malignant bone sarcoma such as malignant osteosarcoma (Osteosarcoma)
  • Ostosarcoma is a bone malignant tumor that occurs in adolescents and children.
  • the tumor is completely resected and the limb is reconstructed (During the surgery, we have to perform wide tumor resection and following limb reconstruction).
  • the way of limb reconstruction includes tumor prosthesis or biological reconstruction.
  • biological reconstruction can achieve better and longerevity limb function, so it is still more commonly used despite the difficulty and difficulty of surgery.
  • frozen autograft uses "real bones" to fill the reconstruct bone defect, but the willingness to donate bones is not high, the source of allogeneic bone is limited, and the bones of each person are included. The size and shape are not the same, so there are defects in the use.
  • malignant osteoma is a tumor that grows from inside or on the bone
  • the bone that grows in the tumor does not necessarily have a serious tumor destruction, even in osteoblastic osteosarcoma. Instead, more bones will grow out of the tumor.
  • the bones are not completely destroyed, so these bones that are full of tumors but still have good texture can be taken out, and the tumor cells are killed by special treatment, and a clean bone is obtained, just like resource recycling. It is called "recycled autograft".
  • the biggest advantage of this method is that it is taken from the bones of the patient itself, without worrying about the size and shape of the bones, and there is no need to worry about disease transmission.
  • the so-called liquid nitrogen freezing autologous bone inactivation is to quickly soak the bone with the tumor in liquid nitrogen such as -196 ° C for a period of time.
  • the central temperature of the tumor can be kept to a minimum temperature and then slowly thawed at room temperature.
  • Tumor cells can be completely killed during rapid freezing and slow thawing.
  • the principle of this cryopreservation is simply to reduce the temperature of the bone with tumor cells to a relatively low temperature, so that water molecules inside and outside the bone cells form crystallization, and these ice crystals destroy the cell membrane and make the tumor cells death.
  • some innocent normal tissues including cartilage, bone and tendon are also destroyed at the same time. Therefore, how to protect normal tissues from being damaged by the freezing process is a problem that has yet to be solved.
  • the present invention is directed to a method of solving the problem of biological tissue being protected from damage during the freezing process.
  • the present invention provides a method for preparing an antifreeze agent, wherein the antifreeze agent is used for protecting a biological tissue from damage during biological tissue freezing treatment, and the preparation method comprises the following steps:
  • the water is physiological saline.
  • the sodium polyacrylate is sodium polyacrylate having a molecular weight of 120,000.
  • the weight ratio of ethylene glycol to water is about 2:1.
  • the weight ratio of the dimethyl sulfoxide to the aqueous ethylene glycol solution is about 1:10.
  • the weight ratio of the substrate to sodium polyacrylate is about 10:1.
  • the substrate prepared in the step (3) is slowly heated and maintained at about 70 °C.
  • a method of preparing an antifreeze according to the present invention comprises the following steps:
  • the substrate prepared in the above step was slowly heated and maintained at about 70 ° C, and sodium polyacrylate was slowly added.
  • the weight ratio of the substrate to the sodium polyacrylate was about 10:1, and the mixture was uniformly cooled to room temperature. Got it.
  • Another aspect of the invention provides an antifreeze which is made by the process of the invention described above.
  • a further aspect of the present invention provides a method for protecting human or animal biological tissues during a freezing process, which uses the antifreezing agent prepared by the above method, is coated on the biological tissue, and is used in the freezing process. Protect the biological tissue from damage.
  • the biological tissue of the human or animal is cartilage, tendon or ligament. In a particular embodiment of the invention, it is used to prepare an autologous bone graft for recycling.
  • an autologous bone graft is obtained, which is obtained by removing an autologous bone with a preparation of a tumor cell from a patient with a bone tumor, and protecting the cartilage, tendon or ligament of the autologous bone with the antifreeze of the present invention, and freezing the same. Treated by killing tumor cells.
  • the osteoma is malignant osteoma.
  • the recovered autogenous bone graft prepared by the method of the present invention can avoid the bone size and shape of the disease due to the bones of the patient itself, and does not cause other diseases to be transmitted, and the freezing treatment kills the tumor cells, With the antifreeze of the present invention, normal biological tissues are not destroyed at the same time.
  • Figure 1 shows an antifreeze made in accordance with the present invention in a transparent viscous form.
  • Fig. 2 shows the protective effect of the antifreeze of the present invention on rat bone tissue.
  • the antifreeze of the present invention can protect cartilage tissue and avoid fragmentation caused by freezing treatment.
  • Fig. 3 shows the effect of the antifreeze of the present invention on apoptosis of rat chondrocytes. Compared with the control group, the antifreeze of the present invention can reduce apoptosis caused by freezing treatment.
  • Figure 4 shows the protective effect of the antifreeze of the present invention on human tissues, relative to the control group,
  • the antifreeze of the invention can reduce the separation of cartilage and hard bone caused by freezing.
  • Figure 5 shows the effect of the antifreeze of the present invention on the change of cartilage color index after freezing. Compared with the control group, the antifreeze of the present invention can alleviate the change of cartilage color index caused by freezing.
  • ethylene glycol 27 g of physiological saline and 10 g of dimethyl sulfoxide (DMSO) were mixed and stirred uniformly to prepare a matrix of antifreeze.
  • 30-70 grams of antifreeze substrate is heated to 70 degrees in a water bath, slowly adding sodium polyacrylate (MW: 120,000) to 5-7 grams, and uniformly cooled to room temperature with stirring, thereby obtaining the antifreeze of the present invention.
  • the appearance is as shown in Figure 1, which is transparent and viscous.
  • the rat knee cartilage was taken out, the control group A was not treated, and the control group B was coated with a conventional commercially available antifreeze (dimethyl sulfoxide) at the articular cartilage.
  • the experimental group was coated with the antifreeze of the present invention at the articular cartilage. They were separately placed in liquid nitrogen for 20 minutes, and then slowly warmed to room temperature for 20 minutes. It was fixed with 4% paraformaldehyde and pathologically sectioned, stained with hematoxylin-eosin (HE stain), control group A (A), control group B (B), experimental group (C) with B.
  • the pathological section of hematoxylin-eosin staining is shown in Figure 2.
  • control group A, control group B, and experimental group C were subjected to apoptosis analysis (TUNEL assay) to verify whether the deoxyribonucleic acid (DNA) of the chondrocytes was prevented from being frozen by the antifreeze of the present invention. Fragmentation, the experimental results are shown in Figure 3. It can be found that the proportion of TUNEL-positive cells (representing apoptosis of the tumor cells) in the experimental group C is significantly lower than that of the control group A and the control group B, indicating that the antifreeze agent of the present invention can reduce apoptosis.
  • TUNEL assay apoptosis analysis
  • the normal cartilage tissue (residual specimens) that were not needed from the human body during surgery was divided into three groups and treated separately (control group A), and traditional commercially available antifreeze was applied to the articular cartilage (control group B). And applying the antifreeze of the present invention to the articular cartilage (experimental group C). They were separately placed in liquid nitrogen for 20 minutes, then slowly warmed to room temperature for 20 minutes at room temperature. The color index change was observed by analyzing the reduction of the brightness of the cartilage surface. The results are shown in Fig. 5. After the control group A and the control group B were frozen, the surface brightness of the cartilage was significantly decreased, indicating that the moisture in the cartilage tissue was largely released after freezing. However, the decrease in the experimental group C was rather small, indicating that the antifreeze of the present invention can alleviate the change in the cartilage color index caused by freezing, and the moisture in the cartilage tissue does not largely escape after freezing.
  • the antifreeze of the present invention can protect the biological tissue from damage caused by freezing during the freezing process.
  • the antifreeze of the present invention can be used as a biological tissue protection during the freezing process.

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Abstract

一种保护生物组织在冷冻处理过程中不受破坏的防冻剂及其制备方法。防冻剂的制备方法包含将乙二醇、水及二甲亚砜三者均匀混合制成基质，再缓慢加入聚丙烯酸钠而得。所制得的抗冻剂在冷冻处理过程中涂覆于该生物组织，可减少生物组织的破坏。

Description

冷冻处理中保护生物组织的防冻剂及其制备方法 技术领域
本发明关于一种防冻剂及其制备方法，特别是用于人体或动物组织冷冻处理过程中保护组织之用。
背景技术
性骨瘤(malignant bone sarcoma)如恶性骨肉瘤(Osteosarcoma)，是青少年与儿童好发的骨头恶性肿瘤。在恶性骨瘤手术治疗的过程中，系先将肿瘤完整切除，并将肢体进行重建(During the surgery,we have to perform wide tumor resection and following limb reconstruction)。而重建(limb reconstruction)的方式包括肿瘤型人工关节(tumor prosthesis)或是生物性重建(Biological reconstruction)。相对于肿瘤型人工关节，生物性重建可达到较佳且恒久(longevity)的肢体功能(limb function)，因此尽管手术困难度较高、较为耗时，仍是较常被使用的方式。其中冷冻自体骨灭活(frozen autograft)，是使用「真正的骨头」来填补骨缺损(reconstruct bone defect)，但因民众愿意捐赠骨头之意愿不高，异体骨来源有限，加上每个人的骨头大小形状不尽相同，因此使用上有其缺陷。
恶性骨瘤虽是由骨头里面或是表面所长出的肿瘤，但是一般而言，长出肿瘤的骨头，肿瘤的破坏并不一定是相当严重，甚至在部分成骨型(osteoblastic)的骨肉瘤当中，反而会因为肿瘤而长出更多的骨头。对于这些病患，其骨头并没有完全被破坏，因此可以将这些长满肿瘤但质地仍不错的骨头取出，经特殊处理将肿瘤细胞杀死，而得到一块干净的骨头，就像资源回收一样，称之为「回收自体移植物(recycled autograft)」。这种方式最大的优点，在于取自病患本身的骨头，不用担心骨头的大小形状不同，也不用担心会有疾病的传染(disease transmission)。而所谓的液态氮冷冻自体骨灭活，即是将带着肿瘤的骨头迅速的浸泡在例如-196℃的液态氮(liquid nitrogen)一段时间，足以确 保肿瘤的中心温度可以达到最低温，再放在室温下缓慢解冻。在此急速冷冻与缓慢解冻的过程中，可将肿瘤细胞完全杀死。这项冷冻处理的原理，简单的说就是藉由将带有肿瘤细胞的骨头温度降到相当低温，使得骨细胞内外的水分子形成冰晶结构(crystallization)，而这些冰晶会摧毁细胞膜而使得肿瘤细胞死亡。然而，冷冻处理过程中，周遭有部分正常的组织(innocent normal tissues)包括软骨(cartilage)、骨头(bone)与肌腱(tendon)也会同时被破坏掉。因此如何保护正常组织避免因冷冻处理过程而遭破坏，为迄今尚待解决的问题。
发明内容
本发明即在提供一种解决生物组织在冷冻处理过程中避免遭破坏的方法。
本发明一方面提供一种制备抗冻剂之方法，其中该抗冻剂系用于生物组织冷冻处理过程中保护生物组织不受破坏，其制备方法包含下列步骤：
(1)取重量比约1～3:1之乙二醇(ethylene glycol)及水，再取二甲基亚砜(dimethyl sulfoxide)，以与上述乙二醇水溶液重量比约1～2:10之比例，将三者均匀混合制成基质；
(2)将上述步骤制得的基质缓慢加热，并加入聚丙烯酸钠(sodium polyacrylate)，其中该基质与聚丙烯酸钠之重量比为4～10:1，搅拌均匀冷却至室温而得。
于本发明之一具体实施例中，该水为生理食盐水。
于本发明之一具体实施例中，该聚丙烯酸钠为具分子量120000之聚丙烯酸钠。
于本发明之一具体实施例中，该乙二醇及水之重量比为约2:1。
于本发明之一具体实施例中，该二甲基亚砜与乙二醇水溶液之重量比为约1:10。
于本发明之一具体实施例中，该基质与聚丙烯酸钠之重量比为约10:1。
于本发明之一具体实施例中，其中该步骤(3)中将制得的基质缓慢加热并维持在约70℃。
根据本发明之一实施例，本发明制备抗冻剂之方法，包含下列步骤：
(1)取重量比约2:1之乙二醇(ethylene glycol)及水，再取二甲基亚砜(dimethyl sulfoxide)，以与上述乙二醇水溶液重量比约1:10之比例，将三者均匀混合制成基质；
(2)将上述步骤制得的基质缓慢加热并维持在约70℃，缓慢加入聚丙烯酸钠(sodium polyacrylate)，该基质与聚丙烯酸钠之重量比为约10:1，搅拌均匀冷却至室温而得。
本发明另一方面提供一种抗冻剂，其系由上述本发明方法制得。
本发明再一方面提供一种在冷冻处理过程中保护人体或动物生物组织之方法，其系使用由上述方法制得的抗冻剂，涂覆在该生物组织上，于冷冻处理过程中用以保护该生物组织减少破坏。
于本发明之一具体实施例中，该人体或动物之生物组织为软骨、肌腱或韧带。于本发明之一特定实施例中，其系用于制备回收自体骨移植物。
本发明又一方面提供一种回收自体骨移植物，其系自骨瘤患者取下带有制备肿瘤细胞的自体骨，以本发明抗冻剂保护该自体骨的软骨、肌腱或韧带，予以冷冻处理杀死肿瘤细胞而得。
于本发明之一具体实施例中，该骨瘤为恶性骨瘤。
依本发明方法制得的回收自体骨移植物，因取自病患本身的骨头，可免骨头大小及形状不同之忧，也不会致其他疾病传染，而冷冻处理杀死肿瘤细胞时，因有本发明抗冻剂保护而正常的生物组织不会同时被破坏掉。
附图说明
图1显示依本发明制成之抗冻剂，呈透明黏稠状。
图2显示本发明抗冻剂对于大鼠骨组织的保护效果，相对于对照组，本发明抗冻剂可保护软骨组织，避免因冷冻处理造成碎裂。
图3显示本发明抗冻剂对于大鼠软骨组织细胞凋亡的影响，相对于对照组，本发明抗冻剂可减少因冷冻处理造成之细胞凋亡。
图4显示本发明抗冻剂对人类组织之保护作用，相对于对照组， 本发明抗冻剂可减少因冷冻造成软骨与硬骨之分离。
图5显示本发明抗冻剂对于冷冻后软骨颜色指数变化之影响，相对于对照组，本发明抗冻剂可减缓因冷冻造成软骨颜色指数之变化。
具体实施方式
实施例1本发明抗冻剂之制备
于室温下取乙二醇(ethylene glycol)63克、生理食盐水27克及二甲基亚砜(Dimethyl sulfoxide，简称DMSO)10克三者混合搅拌均匀，作成抗冻剂基质，取上述所得之抗冻剂基质30-70克于水浴加热维持到70度，缓慢加入聚丙烯酸钠(sodium polyacrylate,MW:120000)至5-7克，搅拌均匀冷却至室温，而得本发明抗冻剂，其外观如图1所示，呈透明黏稠状。
实施例2本发明抗冻剂对于大鼠骨组织的保护效果
将大鼠的膝软骨取出，对照组A不做处理，对照组B在关节软骨处涂上传统一般市售抗冻剂(二甲基亚砜)。实验组在关节软骨处涂上本发明抗冻剂。分别将其置入液态氮中冷冻处理20分钟，接着置于室温下缓慢回温20分钟。再以4％多聚甲醛(paraformaldehyde)固定，并做病理切片，以乙苏木素-伊红染色(HE stain)，对照组A(A)，对照组B(B)，实验组(C)以乙苏木素-伊红染色之病理切片如图2所示。在冷冻过后之对照组A，软骨表面变得脆裂与凹凸不平(箭头处)，对照组B也是凹凸不平(箭头处)，然而在实验组C中，可以见到完整的软骨表面(箭头处)，显示本发明抗冻剂可保护软骨组织，避免因抗冻剂处理的碎裂。
实施例3本发明抗冻剂对于大鼠软骨组织细胞凋亡的影响
将上述对照组A、对照组B、实验组C，分别进行细胞凋亡分析(TUNEL assay)，验证是否可藉由本发明抗冻剂避免因冷冻而造成软骨细胞之脱氧核醣核酸(DNA)之脆裂(fragmentation)，实验结果如图3。可发现实验组C中TUNEL阳性之细胞(代表种肿瘤细胞凋亡)之比例，相较对照组A与对照组B明显较低，显示本发明抗冻剂可减少细胞凋亡。
实施例4本发明抗冻剂对于人体软骨组织的保护作用
将手术中从人体取下不需要的正常软骨组织(残余标本)，分成三组，分别予以不做处理(对照组A)，在关节软骨处涂上传统市售抗冻剂(对照组B)及在关节软骨处涂上本发明抗冻剂(实验组C)。分别将其置入液态氮中冷冻20分钟，接着置于室温下缓慢回温20分钟。再以4％多聚甲醛固定，并做病理切片与苏木素-伊红染色。结果如图4所示，在冷冻过后之对照组A，软骨底部与硬骨相交接触面(星状处)发生分离(separation)，对照组B也发生分离(星状处)，然而在实验组C中，可以见到软骨底部与硬骨相交接触面是正常连结在一起。而此分离的大小，在对照组A为2.16mm,在对照组B为1.2mm，在实验组为0.45mm。显示本发明抗冻剂可保护软骨组织减少因冷冻造成软骨与硬骨之分离。
实施例5本发明抗冻剂对于冷冻后软骨颜色指数变化之影响
将手术中从人体取下不需要的正常软骨组织(残余标本)，分为三组分别予以不做处理(对照组A)，在关节软骨处涂上传统市售抗冻剂(对照组B)及在关节软骨处涂上本发明抗冻剂(实验组C)。分别将其置入液态氮中冷冻20分钟，接着置于室温下缓慢回温20分钟。分析软骨表面亮度之减低(decrease of luminance level)观察其颜色指数变化(color indices change)。结果如图5所示，对照组A与对照组B冷冻过后，软骨之表面亮度明显下降，表示软骨组织内水分在冷冻过后大量脱出。然而在实验组C的下降幅度相当小，显示本发明抗冻剂可减缓因冷冻造成软骨颜色指数之变化，软骨组织内水分在冷冻过后不会大量脱出。
综上，本发明抗冻剂可保护生物组织在冷冻处理过程中因冷冻所造成的伤害。显然，本发明抗冻剂可在冷冻处理过程中作为保护生物组织之用。

Claims (11)

  1. 一种制备抗冻剂之方法，其中该抗冻剂系用于生物组织冷冻处理过程中保护生物组织不受破坏，其制备方法包含下列步骤：
    (1)取重量比约1～3:1之乙二醇(ethylene glycol)及水，再取二甲基亚砜(dimethyl sulfoxide)，以与上述乙二醇水溶液重量比约1～2:10之比例，将三者均匀混合制成基质；
    (2)将上述步骤制得的基质缓慢加热，并加入聚丙烯酸钠(sodium polyacrylate)，其中该基质与聚丙烯酸钠之重量比为4～10:1，搅拌均匀冷却至室温而得。
  2. 根据权利要求1之方法，其中该水为生理食盐水。
  3. 根据权利要求1之方法，其中该聚丙烯酸钠为具分子量120000之聚丙烯酸钠。
  4. 根据权利要求1之方法，其中该乙二醇及水之重量比为约2:1。
  5. 根据权利要求1之方法，其中该二甲基亚砜与乙二醇水溶液之重量比为约1:10。
  6. 根据权利要求1之方法，其中该其中该步骤(3)中将制得的基质缓慢加热并维持在约70℃。
  7. 根据权利要求1之方法，其包含下列步骤：
    (1)取重量比约3:7之乙二醇(ethylene glycol)及水，再取二甲基亚砜(dimethyl sulfoxide)，以与上述乙二醇水溶液重量比约1:10之比例，将三者均匀混合制成基质；
    (2)将上述步骤制得的基质缓慢加热并维持在约70℃，缓慢加入聚丙烯酸钠(sodium polyacrylate)，该基质与聚丙烯酸钠之重量比为约10:1，搅拌均匀冷却至室温而得。
  8. 一种抗冻剂，其系由权利要求1至8之任一项所述之方法制得。
  9. 一种在冷冻处理过程中保护人体或动物生物组织之方法，其将由权利要求1至7之任一项所述之方法制得的抗冻剂涂覆在该生物组织上，于冷冻处理过程中用以保护该生物组织减少破坏。
  10. 根据权利要求9之方法，其中该人体或动物之生物组织为软骨、肌腱或韧带。
  11. 一种回收自体骨移植物，其系自骨瘤患者取下带有制备肿瘤细胞的自体骨，以由权利要求1至7之任一项所述之方法制得的抗冻剂保护该自体骨，予以冷冻处理杀死肿瘤细胞而得。
PCT/CN2016/101112 2016-09-30 2016-09-30 冷冻处理中保护生物组织的防冻剂及其制备方法 WO2018058541A1 (zh)

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