WO2021057234A1 - 一种高强度植入级骨材料的制备方法 - Google Patents

一种高强度植入级骨材料的制备方法 Download PDF

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WO2021057234A1
WO2021057234A1 PCT/CN2020/104732 CN2020104732W WO2021057234A1 WO 2021057234 A1 WO2021057234 A1 WO 2021057234A1 CN 2020104732 W CN2020104732 W CN 2020104732W WO 2021057234 A1 WO2021057234 A1 WO 2021057234A1
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treatment
bone
activated water
treatment liquid
preparation
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French (fr)
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梁新杰
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中新棠国业(苏州)医疗科技有限公司
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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
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention relates to the technical field of bone transplantation, in particular to a method for preparing a high-strength implant-grade bone material.
  • bone transplantation is a common treatment method. According to the materials of bone transplantation, it can be divided into autologous bone transplantation, allogeneic bone transplantation and xenogeneic bone transplantation. Among them, autologous bone and allogeneic bone are limited, but xenogeneic bone is easy to obtain and has a wide range of sources. A natural scaffold material similar in structure to human bone has osteoconductivity and osteoinductive activity. The use of xenogeneic bone as a bone graft material can solve the problem of limited sources of homogenous bone.
  • Xenogeneic bone has clear immunogenicity.
  • the preparation of xenogeneic bone material is mainly the treatment of xenogeneic bone, which reduces or removes the antigenic components in xenogeneic bone and reduces the rejection caused by xenogeneic bone transplantation.
  • conventional xenogeneic bone graft material processing methods require high-strength chemical reagents (sodium hydroxide, etc.) to be treated for a long time to remove high concentrations of xenogeneic cells and strong antigen components in the material, but they are also processed at the same time. It will destroy the collagen and mineral structure in the bone material, and most of the final bone material has low strength and insufficient weight bearing.
  • the purpose of the present invention is to provide a method for preparing high-strength implant-grade bone material.
  • the method of the present invention is used to process heterogeneous bone without damaging the protein structure and minerals.
  • the obtained implant-grade bone material has higher strength.
  • a preparation method of high-strength implant-grade bone material includes the following steps:
  • the first treatment solution includes plasma activated water and a surfactant
  • the second treatment solution includes plasma activated water and hydrogen peroxide
  • the third treatment solution includes plasma activated water and alcohol;
  • the fourth treatment liquid is used to perform the fourth treatment on the heterogeneous bone after the third treatment to obtain a high-strength implant-grade bone material; the fourth treatment liquid includes plasma activated water and A-GAL protease.
  • the mass concentration of the surfactant in the first treatment liquid is 0.1-0.4%;
  • the surfactant includes one or more of Triton reagent, sodium deoxycholate and sodium dodecyl sulfonate.
  • the time of the first treatment is 0.5-2h.
  • the mass concentration of hydrogen peroxide in the step (2) is less than or equal to 1%; the volume ratio of the plasma activated water to the hydrogen peroxide is 1: (0.5-1).
  • the time of the second treatment is 0.5-2h.
  • the volume concentration of alcohol in the step (3) is 65-75%; the volume ratio of the plasma activated water to the alcohol is 1:(0.5-1).
  • the time of the third treatment is 0.5-2h.
  • the mass concentration of A-GAL protease in the fourth treatment solution is 5-15 U/mL.
  • the time of the fourth treatment is 0.5 to 1.5 h.
  • the ratio of the volume of plasma activated water in the first treatment liquid, the second treatment liquid, the third treatment liquid, and the fourth treatment liquid to the mass of the xenogeneic bone is independently 3-10 mL:1g.
  • the present invention provides a method for preparing a high-strength implant-grade bone material, which uses a first treatment liquid including plasma activated water and a surfactant, a second treatment liquid including plasma activated water and hydrogen peroxide, including plasma activated water and The third treatment solution of alcohol and the fourth treatment solution including plasma activated water and A-GAL protease process the xenogeneic bone, and after the treatment, a high-strength implant-grade bone material is obtained.
  • the method provided by the present invention uses plasma activated water to provide a highly active molecular environment and medium, which can catalyze the removal of tissue cells and antigen components, and effectively reduce the damage to the heterogeneous bone protein structure and minerals by chemical reagents such as acid-base reagents. , Maintaining the high mechanical strength of the heterogeneous bone, and at the same time reaching the standard of surgical implantation; further, the preparation time of the method provided by the present invention is significantly shortened, and can be shortened by several days compared with the traditional heterogeneous bone cleaning process.
  • the results of the examples show that the strength of the bone material prepared by the method of the present invention can reach 21.4 ⁇ 3.8 MPa, while the strength of the bone material after the traditional method is only 3.5 ⁇ 1.2 MPa, and the bone material obtained by the preparation method of the present invention is immunogenic. There is no significant difference between the test and the negative control.
  • Figure 1 is an optical microscope image of fresh bovine bone after staining and sectioning
  • Figure 2 is an optical microscope image of the stained section of the bone material treated in Comparative Example 1;
  • Example 3 is an optical microscope image of the stained section of the bone material processed in Example 1;
  • Figure 4 shows the lymphocyte reduction rate of the positive control group, the negative control group and the experimental group in the immunogenicity analysis.
  • the present invention provides a method for preparing a high-strength implant-grade bone material, which includes the following steps:
  • the first treatment solution includes plasma activated water and a surfactant
  • the second treatment solution includes plasma activated water and hydrogen peroxide
  • the third treatment solution includes plasma activated water and alcohol;
  • the fourth treatment liquid is used to perform the fourth treatment on the heterogeneous bone after the third treatment to obtain a high-strength implant-grade bone material; the fourth treatment liquid includes plasma activated water and A-GAL protease.
  • the present invention does not have special requirements for the xenogeneic bone, and the xenogeneic bone commonly used in bone transplantation can be processed by the method of the present invention, such as pig bone, bovine bone or sheep bone.
  • the plasma activated water is preferably obtained by plasma treatment of water; the water is preferably deionized water; the present invention preferably uses a plasma radio frequency electric field to perform plasma treatment on the water; The time is preferably 1 to 5 minutes, and the power is preferably 60 to 80 W; the present invention has no special requirements on the device for generating a plasma radio frequency electric field, and a device well known to those skilled in the art can be used.
  • a large amount of active substances hydroxyl radicals, hydrogen radicals, etc.
  • the present invention uses the characteristic of higher plasma free radical content to provide highly active molecular effects for the treatment process of xenogeneic bone
  • the environment accelerates the reaction process and catalyzes the removal of tissue cells and antigen components of heterogeneous bone.
  • the present invention uses the first treatment liquid to perform the first treatment on the xenogeneic bone to obtain the xenogeneic bone after the first treatment.
  • the first treatment liquid includes plasma activated water and a surfactant; the mass concentration of the surfactant in the first treatment liquid is preferably 0.1-0.4%, more preferably 0.1-0.2%;
  • the surfactant preferably includes one or more of Triton reagent, sodium deoxycholate and sodium dodecyl sulfonate;
  • the Triton reagent preferably includes Triton X-100 (polyethylene glycol octyl phenyl ether) or TritonX-114 (polyoxyethylene mono-tert-octyl phenyl ether);
  • the first treatment time is preferably 0.5-2h, more preferably 0.5-1h, most preferably 0.5h; in the present invention, the surface
  • the active agent plays a role in removing the surface lipids and cell antigens of xenogeneic
  • the present invention uses the second treatment solution to perform the second treatment on the xenogeneic bone after the first treatment to obtain the xenogeneic bone after the second treatment.
  • the second treatment liquid includes plasma activated water and hydrogen peroxide; the mass concentration of the hydrogen peroxide is preferably ⁇ 1%, more preferably 0.3 to 1%, and further preferably 0.3%; the plasma activated water and The volume ratio of hydrogen peroxide is preferably 1:(0.5-1), more preferably 1:(0.6-1), and still more preferably 1:1; the second treatment time is preferably 0.5-2h, more preferably 0.5- 1h, most preferably 0.5h.
  • the hydrogen peroxide plays a role in removing antigen components, and the concentration of hydrogen peroxide used in the present invention is low, the treatment time is short, and the antigen components are effectively removed without destroying the collagen and minerals in the xenogeneic bone. , Which can retain high bone strength.
  • the present invention uses the third treatment liquid to perform the third treatment on the second treatment xenogeneic bone to obtain the third treatment xenogeneic bone.
  • the third treatment liquid includes plasma activated water and alcohol; the volume concentration of the alcohol is preferably 65-75%, more preferably 70%; the volume ratio of the plasma activated water to the alcohol is preferably 1:(0.5-1), more preferably 1:(0.6-1), still more preferably 1:1.
  • the present invention eliminates the reagent residues in the first treatment and the second treatment through the third treatment.
  • the present invention uses the fourth treatment solution to perform the fourth treatment on the third-treated xenogeneic bone to obtain a high-strength implant-grade bone material.
  • the fourth treatment liquid includes plasma activated water and A-GAL protease; the mass concentration of A-GAL protease in the fourth treatment liquid is preferably 5-15 U/mL, more preferably 5 U/mL ;
  • the fourth treatment time is preferably 0.5 to 1.5h, more preferably 0.5 to 1h, most preferably 0.5h; in the present invention, A-GAL protease can remove the A-GAL antigen in xenogeneic bone, further reducing Rejection during xenogeneic bone transplantation.
  • the present invention can complete the elimination of A-GAL antigen in a relatively short time.
  • Traditional xenogeneic bone treatment In the process, it is generally necessary to use A-GAL protease to treat the heterogeneous bone for 72 hours.
  • the method of the present invention greatly shortens the treatment time and reduces the damage to the tissue.
  • the high-strength implant-grade bone material of the present invention is obtained, and the compressive strength of the high-strength implant-grade bone material obtained in the present invention can reach 21.4 ⁇ 3.8 MPa.
  • the ratio of the volume of plasma activated water in the first treatment liquid, the second treatment liquid, the third treatment liquid, and the fourth treatment liquid to the mass of the xenogeneic bone is preferably 3-10 mL:1g, more preferably 4 to 6 mL: 1 g, more preferably 5 mL: 1 g.
  • the present invention has no special requirements on the specific methods of the first treatment, second treatment, third treatment and fourth treatment, and the heterogeneous bone can be treated by immersing the heterogeneous bone in the treatment liquid.
  • the xenogeneic bones used in the following examples and comparative examples are all bovine bones.
  • Plasma treatment of deionized water using plasma radio frequency electric field the treatment time is 2min, the power is 70W; the plasma activated water is obtained; the plasma activated water is used to prepare the treatment solution to treat the bovine bones, the steps are as follows:
  • the ratio of the volume of plasma activated water to the mass of bovine bone is 5mL:1g.
  • step (1) is replaced with sodium deoxycholate.
  • step (1) The other steps are the same as in Example 1, except that the mass percentage of sodium dodecyl sulfonate in step (1) is 0.4%.
  • step (2) The other steps are the same as in Example 1, except that the mass concentration of hydrogen peroxide in step (2) is changed to 0.3 wt.%.
  • the xenogeneic bone was treated by conventional methods in the art.
  • bovine bones of the same position and size as in Example 1 were used for treatment, and the water used for preparing the treatment solution was deionized water.
  • the specific treatment steps are as follows:
  • HE staining method hematoxylin-eosin staining method
  • Figures 1 to 3 The results obtained are shown in Figures 1 to 3, in which Figure 1 is an optical microscope image of fresh bovine bone; Figure 2 is an optical microscope image of the bone material obtained in Comparative Example 1, and Figure 3 is an optical microscope image of the bone material obtained in Example 1. According to Figures 1 to 3, it can be seen that compared with fresh bovine bones, the method in Comparative Example 1 (traditional method) removed most of the xenogeneic cells, but some remained, while the xenogeneic cells in the bone material obtained in Example 1 were basically Completely removed.
  • Example 1 28 ⁇ 1.6 21.4 ⁇ 3.8
  • Example 2 28 ⁇ 3.1 21.2 ⁇ 2.5
  • Example 3 27 ⁇ 1.6 20.5 ⁇ 1.2
  • Example 4 28 ⁇ 3.4 20.1 ⁇ 3.0
  • Example 5 25 ⁇ 2.2 18.4 ⁇ 2.1
  • Example 6 26 ⁇ 3.3 19.2 ⁇ 1.1
  • Example 7 25 ⁇ 1.6 18.1 ⁇ 1.8 Comparative example 1 27 ⁇ 3.6 3.5 ⁇ 1.2
  • the lymphocyte transformation experiment can reflect the level of cellular immunity of the body.
  • the bone material prepared in Example 1 and Comparative Example 1 was co-cultured with human lymphocytes in vitro.
  • the level of lymphocyte transformation rate was detected to show whether the tissue material can cause changes in lymphocytes, so as to prove whether there is a certain amount of bone material. Immunogenicity.
  • Main instruments and reagents microplate reader, 96-well plate centrifuge, lymphocyte counter, AlamarBlue, phytohemagglutinin, lymphocyte separator, fetal bovine serum, L-glutamine, PBS buffer;
  • control and experimental materials prepare control and experimental materials as required (both are powdered samples with a particle size of 200 meshes, obtained by crushing the processed bone material) for use; weigh and pack in a sterile ultra-clean bench;
  • Experimental grouping The experiment is divided into a positive control group, a negative control group and an experimental group.
  • the positive control group is phytohemagglutinin
  • the negative control group is the bone material prepared in Comparative Example 1
  • the experimental group is the bone material prepared in Example 1.
  • Lymphocyte separation add 10 pieces of 4mL fresh anticoagulated whole blood containing different individuals to a centrifuge tube containing 4mL normal saline and mix them, and then add the diluted whole blood to the same amount (4mL) of lymphocytes. Centrifuge the separation solution in the centrifuge tube at 800g for 30 minutes at 20°C, aspirate the lymphocytes in the middle layer and place them in 10 centrifuge tubes with a labeled number, add PBS buffer to 10 mL, mix well, centrifuge at 540g for 10 minutes, and discard The supernatant and the bottom layer are lymphocytes.
  • RPMI1640 complete culture medium containing 10% fetal bovine serum to each tube to 1.5 mL, mix the suspended cells, stain with 4g/L trypan blue, and determine that the cell viability reaches more than 95%. Take 100 ⁇ L of cell suspension and add it to a sample cup with 10mL electrolyte solution. Use a cell counter to count. After counting, the concentration of the original cell suspension is adjusted to 2 ⁇ with RPMI1640 complete culture medium containing 10% fetal bovine serum. 10 9 L -1 , to be used.
  • Lymphocyte transformation experiment 1 Positive control group: mixed culture of human lymphocytes and phytohemagglutinin.
  • 2Negative control group mixed culture of human lymphocytes and bone material obtained in Comparative Example 1.
  • 3Experimental group mixed culture of human lymphocytes and bone material obtained in Example 1;
  • Material mixing Add 10% fetal bovine serum and 10g/L glutamine to RPMI1640 culture medium with a volume fraction of 10% fetal bovine serum and 10g/L glutamine into the weighed experimental sample (ie the bone material prepared in Example 1 and Comparative Example 1), shaker Mix well so that the concentration of bone material is 2g/L respectively, and shake it with a shaker before use to make it completely dispersed evenly;
  • Inoculation Add test material suspension (2g/L) and phytohemagglutinin (0.18g/L) into each horizontal row of 96-well plates according to the above groups, 100 ⁇ L each; then add them in sequence to prepare A good 100 ⁇ L of lymphocyte suspension of different samples, each group of each sample has 3 multiple wells, after adding the plate, the final concentration of the experimental sample in the plate is 1g/L, and the final concentration of phytohemagglutinin is 0.09 g/L; incubate at 37°C in a 5% CO 2 incubator for 48 hours, then add 20 ⁇ L of Alamarblue to each well except the control well, and continue to incubate for 24 hours.
  • the absorbance value measured by the microplate reader After the completion of the culture, take the suspension of each well in the centrifuge at 3000r/min, 4°C, centrifuge for 10min, take 150 ⁇ L of the supernatant and transfer to the corresponding position of the new 96-well plate, and then transfer it to the microplate reader at 570nm , Read the absorbance value at 600nm dual wavelength to calculate the cell reduction rate (ie AlamarBlue reduction rate), which represents the lymphocyte transformation rate, so as to analyze the lymphocyte proliferation.
  • the cell reduction rate ie AlamarBlue reduction rate
  • AlamarBlue reduction rate is calculated according to formula I:
  • AlamarBlue reduction rate (%) (A570nm-A600nm ⁇ R) ⁇ 100% Formula I;
  • R (Mab570nm-M570nm)/(Mab600nm-M600nm);
  • A refers to the absorbance value
  • Mab refers to the absorbance value of the culture medium plus AlmarBlue
  • M refers to the absorbance value of the culture medium
  • R is the correction coefficient
  • the statistical results show that the experimental group and the negative control group are significantly different (P ⁇ 0.05), indicating that the method of the present invention can significantly reduce the antigenicity of xenogeneic bone, and the resulting bone material has low immunogenicity and can achieve surgical implantation. Level standard, and the effect is better than traditional methods.
  • the method provided by the present invention uses plasma activated water to provide a highly active molecular environment and medium, which can effectively reduce the damage of chemical reagents such as acid-base reagents to protein structures and minerals, and maintain the high density of the heterogeneous bone scaffold.
  • the mechanical strength can effectively remove the xenogeneic bone surface antigen at the same time, reaching the standard of surgical implantation level, and the preparation time of the method provided by the present invention is significantly shortened, and the preparation efficiency is improved.

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Abstract

一种高强度植入级骨材料的制备方法，属于骨移植技术领域，依次使用包括等离子活化水和表面活性剂的第一处理液、包括等离子活化水和双氧水的第二处理液、包括等离子活化水和酒精的第三处理液和包括等离子活化水和A-GAL蛋白酶的第四处理液对异种骨进行处理，得到高强度植入级骨材料。该处理方法利用等离子活化水提供高活性的分子作用环境和介质，起到催化清除组织细胞及抗原成分的作用，有效减少化学试剂如酸碱试剂等对蛋白质结构和矿物质的破坏，保持了异种骨支架的高力学强度，并同时使其达到外科植入级的标准，能够显著缩短制备时间，较传统的异种骨清洗工艺能够缩短数天。

Description

一种高强度植入级骨材料的制备方法
本申请要求于2019年9月26日提交中国专利局、申请号为201910915445.5、发明名称为“一种高强度植入级骨材料的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及骨移植技术领域，特别涉及一种高强度植入级骨材料的制备方法。
背景技术
针对由外伤、肿瘤等原因引起的骨缺损等情况，骨移植是常用的治疗方法。根据骨移植的材料可分为自体骨移植、同种异体骨移植和异种骨移植，其中自体骨和同种异体骨取材有限，而异种骨容易获得，来源广泛，并且哺乳动物源性异种骨是与人骨组织结构相似的天然支架材料，具有骨传导和骨诱导活性，使用异种骨作为骨移植材料，可以解决同种骨来源受限的问题。
异种骨具有明确的免疫原性，异种骨骨材料的制备主要是异种骨的处理，通过处理减少或去除异种骨中的抗原成分，降低异种骨移植引起的排斥反应。目前，常规的异种骨植骨材料处理方法都需要用到高强度的化学试剂(氢氧化钠等)长时间的处理，以去除材料中高浓度的异种细胞和强抗原成分，但是在处理的同时也会破坏骨材料中的胶原蛋白和矿物质结构，最终所得骨材料大部分都强度较低、承重不足。
发明内容
本发明目的在于提供一种高强度植入级骨材料的制备方法，利用本发明的方法对异种骨进行处理，不会对蛋白质结构和矿物质产生破坏，所得植入级骨材料具有较高的强度。
为了实现上述发明目的，本发明提供以下技术方案：
一种高强度植入级骨材料的制备方法，包括以下步骤：
(1)使用第一处理液对异种骨进行第一处理，得到第一处理后的异种骨；所述第一处理液中包括等离子活化水和表面活性剂；
(2)使用第二处理液对第一处理后的异种骨进行第二处理，得到第 二处理后的异种骨；所述第二处理液中包括等离子活化水和双氧水；
(3)使用第三处理液对第二处理后的异种骨进行第三处理，得到第三处理后的异种骨；所述第三处理液中包括等离子活化水和酒精；
(4)使用第四处理液对第三处理后的异种骨进行第四处理，得到高强度植入级骨材料；所述第四处理液中包括等离子活化水和A-GAL蛋白酶。
优选的，所述第一处理液中表面活性剂的质量浓度为0.1～0.4％；
所述表面活性剂包括Triton试剂、脱氧胆酸钠和十二烷基磺酸钠中的一种或几种。
优选的，所述第一处理的时间为0.5～2h。
优选的，所述步骤(2)中双氧水的质量浓度≤1％；所述等离子活化水和双氧水的体积比为1:(0.5～1)。
优选的，所述第二处理的时间为0.5～2h。
优选的，所述步骤(3)中酒精的体积浓度为65～75％；所述等离子活化水和酒精的体积比为1:(0.5～1)。
优选的，所述第三处理的时间为0.5～2h。
优选的，所述第四处理液中A-GAL蛋白酶质量浓度为5～15U/mL。
优选的，所述第四处理的时间为0.5～1.5h。
优选的，所述第一处理液、第二处理液、第三处理液和第四处理液中等离子活性水的体积和异种骨的质量之比独立地为3～10mL:1g。
本发明提供了一种高强度植入级骨材料的制备方法，依次使用包括等离子活化水和表面活性剂的第一处理液、包括等离子活化水和双氧水的第二处理液、包括等离子活化水和酒精的第三处理液和包括等离子活化水和A-GAL蛋白酶的第四处理液对异种骨进行处理，处理后得到高强度植入级骨材料。本发明提供的方法利用等离子活化水提供高活性的分子作用环境和介质，起到催化清除组织细胞及抗原成分的作用，有效减少化学试剂如酸碱试剂等对异种骨蛋白质结构和矿物质的破坏，保持了异种骨的高力学强度，并同时达到外科植入级的标准；进一步的，本发明提供的方法制备时间显著缩短，较传统的异种骨清洗工艺能够缩短数天。实施例结果表明，本发明的方法制备得到的骨材料强度可达到21.4±3.8MPa，而传统方 法处理后的骨材料强度仅为3.5±1.2MPa，并且本发明制备方法得到的骨材料免疫原性检测与阴性对照无明显差异。
说明书附图
图1为新鲜牛骨染色切片后的光学显微镜图；
图2为对比例1处理后的骨材料染色切片后的光学显微镜图；
图3为实施例1处理后的骨材料染色切片后的光学显微镜图；
图4为免疫原性分析中阳性对照组、阴性对照组和实验组的淋巴细胞还原率。
具体实施方式
下面结合实施例和附图对本发明进行进一步说明。
本发明提供了一种高强度植入级骨材料的制备方法，包括以下步骤：
(1)使用第一处理液对异种骨进行第一处理，得到第一处理后的异种骨；所述第一处理液中包括等离子活化水和表面活性剂；
(2)使用第二处理液对第一处理后的异种骨进行第二处理，得到第二处理后的异种骨；所述第二处理液中包括等离子活化水和双氧水；
(3)使用第三处理液对第二处理后的异种骨进行第三处理，得到第三处理后的异种骨；所述第三处理液中包括等离子活化水和酒精；
(4)使用第四处理液对第三处理后的异种骨进行第四处理，得到高强度植入级骨材料；所述第四处理液中包括等离子活化水和A-GAL蛋白酶。
本发明对所述异种骨没有特殊要求，骨移植常用的异种骨均可使用本发明的方法处理，具体的如猪骨、牛骨或羊骨等。
在本发明中，所述等离子活化水优选通过对水进行等离子体处理后得到；所述水优选为去离子水；本发明优选使用等离子射频电场对水进行等离子体处理；所述等离子体处理的时间优选为1～5min，功率优选为60～80W；本发明对产生等离子射频电场的装置没有特殊要求，使用本领域技术人员熟知的装置即可。对水进行等离子体处理后，水中产生大量的活性物质(羟基自由基、氢自由基等)，本发明利用等离子体自由基含量较高的特点，为异种骨的处理过程提供高活性的分子作用环境，加快反应进程，催化清除异种骨的组织细胞及抗原成分。
本发明使用第一处理液对异种骨进行第一处理，得到第一处理后的异种骨。在本发明中，所述第一处理液中包括等离子活化水和表面活性剂；所述第一处理液中表面活性剂的质量浓度优选为0.1～0.4％，更优选为0.1～0.2％；所述表面活性剂优选包括Triton试剂、脱氧胆酸钠和十二烷基磺酸钠中的一种或几种；所述Triton试剂优选包括TritonX-100(聚乙二醇辛基苯基醚)或TritonX-114(聚氧乙烯单叔辛基苯基醚)；所述第一处理的时间优选为0.5～2h，更优选为0.5～1h，最优选为0.5h；在本发明中，所述表面活性剂起到清除异种骨表面脂质和细胞抗原的作用，并且由于等离子活性水具有较高的活性，能够促进表面活性剂对异种骨表面脂质和细胞抗原的反应，因而在较低浓度下进行短时间的处理即可达到清除效果，减小了对组织的损伤，在本领域的常规异种骨处理工艺中，一般需要使用高浓度表面活性剂处理72h，本发明的方法大大缩短了处理时间。
第一处理完成后，本发明使用第二处理液对第一处理后的异种骨进行第二处理，得到第二处理后的异种骨。在本发明中，所述第二处理液中包括等离子活化水和双氧水；所述双氧水的质量浓度优选≤1％，更优选为0.3～1％，进一步优选为0.3％；所述等离子活性水和双氧水的体积比优选为1:(0.5～1)，更优选为1:(0.6～1)，进一步优选为1:1；所述第二处理的时间优选为0.5～2h，更优选为0.5～1h，最优选为0.5h。在本发明中，所述双氧水起到清除抗原成分的作用，并且本发明使用的双氧水浓度低，处理时间短，在有效清除抗原成分的同时不会对异种骨中的胶原蛋白和矿物质产生破坏，因而可以保留较高的骨强度。
第二处理完成后，本发明使用第三处理液对第二处理后的异种骨进行第三处理，得到第三处理后的异种骨。在本发明中，所述第三处理液中包括等离子活化水和酒精；所述酒精的体积浓度优选为65～75％，更优选为70％；所述等离子活化水和酒精的体积比优选为1:(0.5～1)，更优选为1:(0.6～1)，进一步优选为1:1。本发明通过第三处理清除第一处理和第二处理中的试剂残留。
第三处理完成后，本发明使用第四处理液对第三处理后的异种骨进行第四处理，得到高强度植入级骨材料。在本发明中，所述第四处理液中包括等离子活化水和A-GAL蛋白酶；所述第四处理液中A-GAL蛋白酶的 质量浓度优选为5～15U/mL，更优选为5U/mL；所述第四处理的时间优选为0.5～1.5h，更优选为0.5～1h，最优选为0.5h；在本发明中，A-GAL蛋白酶能够去除异种骨中的A-GAL抗原，进一步降低异种骨移植时的排斥反应。在本发明中，等离子活化水中存在大量不同电荷的自由基，能够促进A-GAL蛋白酶和抗原的作用，因而本发明在较短时间内即可完成A-GAL抗原的清除，传统的异种骨处理工艺中一般需要使用A-GAL蛋白酶对异种骨处理72h，本发明的方法大大缩短了处理时间，减小了对组织的损伤。第四处理完毕后，即得到本发明的高强度植入级骨材料，本发明得到的高强度植入级骨材料抗压强度能够达到21.4±3.8MPa。
在本发明中，所述第一处理液、第二处理液、第三处理液和第四处理液中等离子活性水的体积和异种骨的质量之比优选为3～10mL：1g，更优选为4～6mL：1g，进一步优选为5mL：1g。本发明对所述第一处理、第二处理、第三处理和第四处理的具体方式没有特殊要求，将异种骨浸没在处理液中进行处理即可。
下面结合实施例对本发明提供的方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
下述实施例及对比例中使用的异种骨均为牛骨。
实施例1
使用等离子射频电场对去离子水进行等离子体处理，处理时间为2min，功率为70W；得到等离子活性水；使用等离子活性水配制处理液，对牛骨进行处理，步骤如下：
(1)将等离子活性水和十二烷基磺酸钠混合，控制十二烷基磺酸钠的质量百分含量为0.1％，得到第一处理液，使用第一处理液处理牛骨0.5h；
(2)将等离子活性水和1wt.％的双氧水以体积比1:1混合，得到第二处理液，使用第二处理液处理牛骨0.5h；
(3)将等离子活性水和体积浓度70％的酒精以体积比1:1混合，得到第三处理液，使用第三处理液处理牛骨0.5h；
(4)将等离子活性水和A-GAL蛋白酶混合，得到第四处理液，控制第四处理液中A-GAL蛋白酶的质量浓度为5U/mL，使用第四处理液处 理牛骨0.5h，处理完毕后，得到高强度植入级骨材料；
步骤(1)～(4)中，等离子活性水的体积和牛骨的质量比为5mL：1g。
实施例2
其他步骤和实施例1相同，仅将步骤(1)中的表面活性剂替换为脱氧胆酸钠。
实施例3
其他步骤和实施例1相同，仅将步骤(1)中十二烷基磺酸钠的质量百分含量为0.4％。
实施例4
其他步骤和实施例1相同，仅将步骤(2)中双氧水的质量浓度改为0.3wt.％。
实施例5
其他步骤和实施例1相同，仅将步骤(1)～(4)的处理时间改为1h；
实施例6
其他步骤和实施例1相同，仅将步骤(1)～(4)的处理时间改为1.5h。
实施例7
其他步骤和实施例1相同，仅将步骤(1)～(3)的处理时间改为2h。
对比例1
采用本领域常规方法处理异种骨，实验中使用和实施例1相同部位、相同尺寸的牛骨进行处理，配制处理液所用水为去离子水，具体处理步骤如下：
将牛骨处理浸泡在十二烷基磺酸钠溶液(质量浓度为1％)处理72h(脱脂)；使用双氧水溶液(双氧水质量浓度为30％)处理8h(脱蛋白)，最后使用A-GAL蛋白酶液(浓度为30U/mL)处理72h(脱A-GAL)，得到处理后的异种骨。
细胞残留测试
使用苏木素-伊红染色法(HE染色法)对新鲜牛骨、实施例1处理后的牛骨和对比例1处理后的牛骨进行处理，主要处理步骤如下：
(1)将标本浸没于10％甲醛溶液中(配方如下：36％甲酸溶液25mL，蒸馏水225mL)72小时；
(2)1mol/L盐酸溶液脱钙12小时；
(3)使用乙醇逐级脱水，然后进行二甲苯透明和石蜡包埋；
(4)不连续切片、苏木素-伊红染色；
(5)光学显微镜下观察，拍照。
所得结果如图1～3所示，其中图1为新鲜牛骨的光学显微镜图；图2为对比例1所得骨材料的光学显微镜图，图3为实施例1所得骨材料的光学显微镜图。根据图1～3可以看出：和新鲜牛骨相比，对比例1中的方法(传统方法)去除了大部分异种细胞，但仍有部分残留，而实施例1所得骨材料中异种细胞基本被完全去除。
对实施例2～7所得骨材料进行相同的检测，所得结果和实施例1相似，异种细胞基本被完全去除。
抗压强度测试：
使用RGT-20A微机控制电子万能材料试验机检测样品的抗压性能，试验条件为室温，湿度为30％，加载速度为5mm/min，试样尺寸为5mm*5mm*5mm，每组5个试样，分别测试实施例1～7及对比例1处理前后牛骨的抗压强度，实验结果用
Figure PCTCN2020104732-appb-000001
表示，所得结果如表1所示：
表1实施例1～7及对比例1处理前后牛骨强度
项目 处理前牛骨抗压强度(MPa) 处理后牛骨抗压强度(MPa)
实施例1 28±1.6 21.4±3.8
实施例2 28±3.1 21.2±2.5
实施例3 27±1.6 20.5±1.2
实施例4 28±3.4 20.1±3.0
实施例5 25±2.2 18.4±2.1
实施例6 26±3.3 19.2±1.1
实施例7 25±1.6 18.1±1.8
对比例1 27±3.6 3.5±1.2
根据表1可以看出，实施例1～7处理后的骨材料均保持了较高的力学强度，而对比例1处理后的骨材料抗压强度仅为3.5±1.2MPa，强度大 幅降低。
免疫原性分析
淋巴细胞转化实验作为测定机体免疫功能的重要实验，可以反映机体的细胞免疫水平。实验采用实施例1和对比例1制备的骨材料与人的淋巴细胞体外共同培养，通过检测淋巴细胞转化率的高低来说明组织材料能否引起淋巴细胞的变化，从而证明骨材料是否存在一定的免疫原性作用。
主要仪器及试剂：酶标仪、96孔板离心机、淋巴细胞计数仪、AlamarBlue、植物血球凝集素、淋巴细胞分离液、胎牛血清、L-谷氨酰胺、PBS缓冲液；
实验材料的准备：按要求准备对照和实验材料(均选用粉状样品，粒径200目，将处理后的骨材料粉碎后得到)待用；在无菌超净台内称质量分装；
实验分组：实验分为阳性对照组、阴性对照组和实验组，其中阳性对照组为植物血球凝集素，阴性对照组为对比例1制备的骨材料，实验组为实施例1制备的骨材料。
淋巴细胞的分离：将10支装有不同个体的4mL新鲜抗凝全血分别加入到装有4mL生理盐水的离心管内混匀，再分别将稀释全血加入到装有等量(4mL)淋巴细胞分离液的离心管内，在800g，20℃下离心30min，吸出中间层的淋巴细胞置于已标记好编号的10支离心管内，并加PBS缓冲液至10mL，混匀，540g离心10min，弃去上清，底层为淋巴细胞。各管内加含体积分数为10％胎牛血清的RPMI1640完全培养液至1.5mL，混匀悬浮细胞，使用4g/L锥虫蓝染色，测定细胞活性达95％以上。分别取100μL细胞悬液加入到有10mL电解液样品杯中，应用细胞计数仪进行计数，计数后将原细胞悬液浓度用含体积分数为10％胎牛血清的RPMI1640完全培养液调整为2×10 9L -1，待用。
淋巴细胞转化实验：①阳性对照组：人淋巴细胞与植物血球凝集素混合培养。②阴性对照组：人淋巴细胞与对比例1所得骨材料混合培养。③实验组：人淋巴细胞与实施例1所得骨材料混合培养；
与淋巴细胞混合培养的具体步骤如下：
材料混匀：向已经称重好的实验样品(即实施例1和对比例1制备的 骨材料)中加入体积分数为10％胎牛血清及10g/L谷氨酰胺的RPMI1640培养液，振荡仪混匀，使骨材料的浓度分别为2g/L，使用前经振荡仪振荡使其完全分散均匀；
接种：按照上述分组在96孔板由上到下每一横排孔内分别加入实验材料悬液(2g/L)，植物血球凝集素(0.18g/L)，各100μL；再分别依次加入制备好的不同样本的淋巴细胞悬液100μL，每个样本的每个组设3个复孔，加板后最终使得板内实验样品的终浓度为1g/L，植物血球凝集素的终浓度为0.09g/L；于37℃、体积分数为5％CO 2温箱中培养48h，再向除对照孔外各孔加入20μL的Alamarblue(阿尔玛蓝)，继续培养24h。
酶标仪测吸光度值：培养完成后取各孔悬液于离心机内3000r/min，4℃，离心10min，分别取上清150μL转移至新的96孔板相应位置后，在酶标仪570nm，600nm双波长下读取吸光度值，以计算细胞还原率(即AlamarBlue还原率)，即代表淋巴细胞转化率，从而分析淋巴细胞增殖的情况。
AlamarBlue还原率按照式I进行计算：
AlamarBlue还原率(％)＝(A570nm-A600nm×R)×100％   式I；
式I中：R＝(Mab570nm-M570nm)/(Mab600nm-M600nm)；
A指吸光度值；Mab指的是培养液加AlmarBlue的吸光度值；M指培养液的吸光度值；R为校正系数。
使用SPSS
Figure PCTCN2020104732-appb-000002
统计软件处理，实验结果用
Figure PCTCN2020104732-appb-000003
表示，两组均数间比较采用两独立样本t检验；多个样本均数之间比较采用方差分析，以P<0.05认为差异有显著性意义；所得结果见图4，相关数据见表2。
表2淋巴细胞还原率
Figure PCTCN2020104732-appb-000004
此外，统计结果显示实验组和阴性对照组相比具有显著性差异(P<0.05)，说明本发明的方法能够显著降低异种骨的抗原性，所得骨材 料免疫原性低，能够达到外科植入级的标准，且效果优于传统方法。
对实施例2～7所得骨材料进行相同的免疫原性检测，所得结果相似，均显示处理后的骨材料具有较低的免疫原性。
由以上实施例可知，本发明提供的方法利用等离子活化水提供高活性的分子作用环境和介质，能够有效减少化学试剂如酸碱试剂等对蛋白质结构和矿物质的破坏，保持异种骨支架的高力学强度，同时能有效去除异种骨表面抗原，达到外科植入级的标准，且本发明提供的方法制备时间显著缩短，提高了制备效率。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种高强度植入级骨材料的制备方法，其特征在于，包括以下步骤：
    (1)使用第一处理液对异种骨进行第一处理，得到第一处理后的异种骨；所述第一处理液中包括等离子活化水和表面活性剂；
    (2)使用第二处理液对第一处理后的异种骨进行第二处理，得到第二处理后的异种骨；所述第二处理液中包括等离子活化水和双氧水；
    (3)使用第三处理液对第二处理后的异种骨进行第三处理，得到第三处理后的异种骨；所述第三处理液中包括等离子活化水和酒精；
    (4)使用第四处理液对第三处理后的异种骨进行第四处理，得到高强度植入级骨材料；所述第四处理液中包括等离子活化水和A-GAL蛋白酶。
  2. 根据权利要求1所述的制备方法，其特征在于，所述第一处理液中表面活性剂的质量浓度为0.1～0.4％；
    所述表面活性剂包括Triton试剂、脱氧胆酸钠和十二烷基磺酸钠中的一种或几种。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述第一处理的时间为0.5～2h。
  4. 根据权利要求1所述的制备方法，其特征在于，所述步骤(2)中双氧水的质量浓度≤1％；所述等离子活化水和双氧水的体积比为1:(0.5～1)。
  5. 根据权利要求1或4所述的制备方法，其特征在于，所述第二处理的时间为0.5～2h。
  6. 根据权利要求1所述的制备方法，其特征在于，所述步骤(3)中酒精的体积浓度为65～75％；所述等离子活化水和酒精的体积比为1:(0.5～1)。
  7. 根据权利要求1或6所述的制备方法，其特征在于，所述第三处理的时间为0.5～2h。
  8. 根据权利要求1所述的制备方法，其特征在于，所述第四处理液 中A-GAL蛋白酶质量浓度为5～15U/mL。
  9. 根据权利要求1或8所述的制备方法，其特征在于，所述第四处理的时间为0.5～1.5h。
  10. 根据权利要求1所述的制备方法，其特征在于，所述第一处理液、第二处理液、第三处理液和第四处理液中等离子活性水的体积和异种骨的质量之比独立地为3～10mL:1g。
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