CN111973807B - 一种仿生人工颞下颌关节盘及其制备方法 - Google Patents

一种仿生人工颞下颌关节盘及其制备方法 Download PDF

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CN111973807B
CN111973807B CN202010896047.6A CN202010896047A CN111973807B CN 111973807 B CN111973807 B CN 111973807B CN 202010896047 A CN202010896047 A CN 202010896047A CN 111973807 B CN111973807 B CN 111973807B
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temporomandibular joint
disc
joint disc
flocculent
room temperature
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祝颂松
姜楠
毕瑞野
王旻
应彬彬
陈浩哲
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Sichuan University
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Abstract

本发明提供了一种仿生人工颞下颌关节盘及其制备方法，制备方法包括以下步骤：收取牛尾的椎间盘，清洗，然后冻融处理，提取糖胺聚糖和蛋白，再打碎至絮状，加入十二烷基硫酸钠和DNA酶室温处理，洗净，得椎间盘絮状样品；将椎间盘絮状样品冷冻干燥，然后加入消化液，共混，调节pH值至中性，再加入糖胺聚糖和蛋白，得生物墨水；将高分子材料和生物墨水在室温下共混，进行3D打印，得仿生人工颞下颌关节盘。本发明还包括采用上述方法制得的仿生人工颞下颌关节盘。本发明以脱细胞椎间盘制得生物墨水，并辅以高分子材料，利用3D打印方法重建颞下颌关节盘，有效解决了现有技术中无法匹配颞下颌关节的生理负荷、重建困难和成本较高等问题。

Description

一种仿生人工颞下颌关节盘及其制备方法
技术领域
本发明属于生物医用材料技术领域，具体涉及一种仿生人工颞下颌关节盘及其制备方法。
背景技术
颞下颌关节盘(Temporomandibular joint disk,TMJ disk)是位于下颌髁突和颞骨关节窝之间的纤维软骨样组织，是颞下颌关节行使功能的主要组成部分和重要基础，主要作用在于润滑关节面、分散载荷、缓冲震荡、减少髁突与关节窝骨性结构之间的不协调。临床上颞下颌关节盘的破坏或缺失是引起颞下颌关节疾病的主要病因之一，进而也会造成整个口颌***功能异常，严重影响患者的生活质量。据相关资料显示，全世界每年需要重建关节盘的患者多达百万例，所需医疗费用也高达数亿美元。
颞下颌关节盘是一个内外径大于前后径的椭圆形纤维软骨盘，其解剖结构与组织特性都是为了满足其生物力学功能的实现。目前，有关关节盘的生物力学研究已经表明：颞下颌关节盘在关节的功能运动中需要承受摩擦、压缩、拉伸和剪切等多个分量的力，因此颞下颌关节盘也是人体最好发穿孔、破裂等疾病的纤维软骨器官。由于缺乏血管和细胞，代谢缓慢，颞下颌关节盘一旦发生缺损，自身修复能力极差。
目前，临床上常用的颞下颌关节盘修复替代材料主要包括脱细胞异体组织补片和自身临近组织瓣膜的转移修复，但无论解剖学结构还是组织学成分，尤其是替代插补物的生物力学性能，都与颞下颌关节盘相去甚远，无法匹配颞下颌关节的生理负荷。同时，颞下颌关节盘形态特殊，成分多样，结构复杂，利用人工材料很难获得效果良好的仿生替代品，这更使颞下颌关节盘重建难上加难。因此，如何寻找或构建一种新的颞下颌关节盘替代材料已经成为颞下颌关节疾病治疗领域中一个亟待解决的重要课题。
发明内容
针对现有技术中存在的上述问题，本发明提供一种仿生人工颞下颌关节盘及其制备方法，以脱细胞椎间盘制得生物墨水，并辅以高分子材料，利用3D打印方法重建颞下颌关节盘，有效解决了现有技术中无法匹配颞下颌关节的生理负荷、重建困难和成本较高等问题。
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：提供一种仿生人工颞下颌关节盘的制备方法，包括以下步骤：
(1)在无菌条件下收取牛尾的椎间盘，清洗，然后在-20～37℃温度区间冻融处理4～6个循环，采用超纯水/盐酸胍化学试剂提取糖胺聚糖和蛋白，再将剩余组织打碎至絮状，加入4～6wt％的十二烷基硫酸钠和4～6KU/ml的DNA酶室温处理2～4h，无酶水洗净，得椎间盘絮状样品；
(2)将步骤(1)所得椎间盘絮状样品冷冻干燥，然后加入消化液，在室温、25～35r/min条件下共混60～84h，调节pH值至中性，再加入步骤(1)所得糖胺聚糖和蛋白，得生物墨水；
(3)将生物相容性好的高分子材料和步骤(2)所得生物墨水按质量比1:1～3在室温下共混，然后进行3D打印，得仿生人工颞下颌关节盘。
进一步，步骤(1)中，在无菌条件下收取8月龄牛尾的椎间盘，用生理盐水清洗去除血迹。
进一步，步骤(1)中，加入5wt％的十二烷基硫酸钠和5KU/ml的DNA酶室温处理3h。
进一步，步骤(2)中，消化液通过以下方法制备得到：将胃蛋白酶溶于0.4～0.6vt％的醋酸溶液中，得消化液；胃蛋白酶与椎间盘絮状样品干重质量比为1:9～11。
进一步，醋酸溶液浓度为0.5vt％，胃蛋白酶与椎间盘絮状样品干重质量比为1:10。
进一步，步骤(2)中，用氢氧化钠调节pH值至中性。
进一步，步骤(3)中，生物相容性好的高分子材料为降冰片烯改性透明质酸或GelMA(甲基丙烯酸化水凝胶)。
进一步，步骤(3)中，高分子材料和生物墨水按质量比1:1在室温下共混。
上述的仿生人工颞下颌关节盘的制备方法制得的仿生人工颞下颌关节盘。
综上所述，本发明具备以下优点：
1、本发明先将椎间盘进行脱细胞处理，去除异种动物椎间盘的免疫原性，然后以脱细胞椎间盘制得生物墨水，并辅以高分子材料增强力学性能，利用3D打印方法重建颞下颌关节盘，该方法方便快捷，制备成本较低，可靠性强，临床转化价值大，便于推广使用，有效解决了现有技术中无法匹配颞下颌关节的生理负荷、重建困难和成本较高等问题。椎间盘与颞下颌关节盘同属纤维软骨，且来源丰富，成本低廉，是实现颞下颌关节盘器官再造是解决前述问题的极佳选择。
2、颞下颌关节盘形态特殊，成分多样，结构复杂，利用人工材料很难获得相似成分、替代效果良好的仿生替代品；但颞下颌关节盘与椎间盘、半月板等组织同属于纤维软骨，基本成分一致，组织学及功能特点亦有类似。由于椎间盘供材丰富，每只动物几十个，来源丰富；利用椎间盘制备成分类似的关节盘就是一个很好的思路。但椎间盘与关节盘形态不一致，因此需要做成生物墨水进行3D打印，仿生形态。
3、在制备时，首先收取牛尾的椎间盘并去除血迹，然后进行脱细胞处理，在过程中提取糖胺聚糖和相关蛋白待用，再将余留的椎间盘组织打碎至絮状，絮状更有利于形成生物墨水，并用十二烷基硫酸钠和DNA酶除去其内的细胞成分，无酶水洗净得椎间盘絮状样品；将絮状样品冷冻干燥去除水分，然后加入胃蛋白酶和醋酸溶液形成的消化液，室温共混并调节至中性，最后加入先前提取的糖胺聚糖和相关蛋白，得生物墨水；将生物墨水和高分子材料(用于增强生物墨水的力学性能)在室温下共混，再通过3D打印的方法进行仿生打印，可以得到仿生人工颞下颌关节盘。该方法以牛尾的椎间盘为原料，并辅以高分子材料，利用3D打印方法重建颞下颌关节盘，其制备方法简单易操作，能够快速制得仿生人工颞下颌关节盘，能够满足现目前的使用要求，且椎间盘却来源广泛、造价低廉，能够有效降低成本，便于推广使用。
附图说明
图1为椎间盘脱细胞处理流程；
图2为脱细胞后的椎间盘制备成生物墨水流程；
图3为脱细胞完成后各组样品DNA含量；
图4为脱细胞完成后各组样品胶原含量；
图5为生物墨水动态弹性模量力学性能测试结果。
其中，图5中较大方形和三角形为生物墨水测试结果，较小方形和三角形为高分子加强后生物墨水(即本申请所得生物墨水)测试结果。
具体实施方式
以下结合实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
一种仿生人工颞下颌关节盘，其制备方法包括以下步骤：
(1)在无菌条件下收取8月龄牛尾的椎间盘，用青霉素生理盐水清洗去除血迹，然后在-20～37℃温度区间冻融处理5个循环，采用超纯水/盐酸胍化学试剂提取糖胺聚糖和蛋白，再将剩余组织打碎至絮状，加入4wt％的十二烷基硫酸钠(SDS)和4KU/ml的DNA酶室温处理2h，无酶水洗净，得椎间盘絮状样品；
(2)将步骤(1)所得椎间盘絮状样品冷冻干燥，然后加入消化液，在室温、25r/min条件下共混60h，用氢氧化钠调节pH值至中性，再加入步骤(1)所得糖胺聚糖和蛋白，得生物墨水；消化液通过以下方法制备得到：将胃蛋白酶溶于0.4vt％的醋酸溶液中，得消化液；胃蛋白酶与椎间盘絮状样品干重质量比为1:9；
(3)将降冰片烯改性透明质酸高分子材料和步骤(2)所得生物墨水按质量比1:1在室温下共混，然后进行3D打印，得仿生人工颞下颌关节盘。
其中，上述所用试剂为SDS(Sigma,Cat#L4390-100G,Batch#096K0041)、DNA酶(Sigma,150KU,D5025-150KU)、Triton-100(Sigma,250ml,T8787-250)、胃蛋白酶(Sigma,P7125-100G)、冰乙酸(Sigma,45726-1L-F)和NaOH(Fluka,71692-1KG)。
实施例2
一种仿生人工颞下颌关节盘，其制备方法包括以下步骤：
(1)在无菌条件下收取8月龄牛尾的椎间盘，用青霉素生理盐水清洗去除血迹，然后在-20～37℃温度区间冻融处理5个循环，采用超纯水/盐酸胍化学试剂提取糖胺聚糖和蛋白，再将剩余组织打碎至絮状，加入5wt％的十二烷基硫酸钠和5KU/ml的DNA酶室温处理5h，无酶水洗净，得椎间盘絮状样品；
(2)将步骤(1)所得椎间盘絮状样品冷冻干燥，然后加入消化液，在室温、30r/min条件下共混72h，用氢氧化钠调节pH值至中性，再加入步骤(1)所得糖胺聚糖和蛋白，得生物墨水；消化液通过以下方法制备得到：将胃蛋白酶溶于0.5vt％的醋酸溶液中，得消化液；胃蛋白酶与椎间盘絮状样品干重质量比为1:10；
(3)将降冰片烯改性透明质酸高分子材料和步骤(2)所得生物墨水按质量比1:1在室温下共混，然后进行3D打印，得仿生人工颞下颌关节盘。
实施例3
一种仿生人工颞下颌关节盘，其制备方法包括以下步骤：
(1)在无菌条件下收取8月龄牛尾的椎间盘，用青霉素生理盐水清洗去除血迹，然后在-20～37℃温度区间冻融处理5个循环，采用超纯水/盐酸胍化学试剂提取糖胺聚糖和蛋白，再将剩余组织打碎至絮状，加入6wt％的十二烷基硫酸钠和6KU/ml的DNA酶室温处理4h，无酶水洗净，得椎间盘絮状样品；
(2)将步骤(1)所得椎间盘絮状样品冷冻干燥，然后加入消化液，在室温、35r/min条件下共混84h，用氢氧化钠调节pH值至中性，再加入步骤(1)所得糖胺聚糖和蛋白，得生物墨水；消化液通过以下方法制备得到：将胃蛋白酶溶于0.6vt％的醋酸溶液中，得消化液；胃蛋白酶与椎间盘絮状样品干重质量比为1:11；
(3)将GelMA(甲基丙烯酸化水凝胶)高分子材料和步骤(2)所得生物墨水按质量比1:1在室温下共混，然后进行3D打印，得仿生人工颞下颌关节盘。
实验例
分别采用单纯5％SDS(Sample after extraction)、5％SDS联合5KU/ml DNA酶(DNAse pretreatment)、5％SDS联合5KU/ml DNA酶联合Triton-100(DNAse pretreatment+0.1％Triton 100)进行脱细胞处理，其余操作方法与实施例2相同。处理结束后，利用定量测量及组织学评估脱细胞效果和残余成分胶原含量以及生物墨水动态弹性模量力学性能测试结果，其结果见图3～5。
由图3可知，单纯5％SDS(Sample after extraction)、5％SDS联合5KU/ml DNA酶(DNAse pretreatment)、5％SDS联合5KU/ml DNA酶联合Triton-100(DNAse pretreatment+0.1％Triton 100)三种处理方式都可以将组织中的DNA含量降至国际脱细胞标准以下(干重含量≤50ng/mg)，满足临床要求。且5％SDS联合5KU/ml DNA酶(DNAse pretreatment)的处理效果最好。
由图4可知，单纯5％SDS(Sample after extraction)、5％SDS联合5KU/ml DNA酶(DNAse pretreatment)、5％SDS联合5KU/ml DNA酶联合Triton-100(DNAse pretreatment+0.1％Triton 100)三种处理方式后脱细胞组织中的胶原含量干重均接近900ug/mg，说明脱细胞后的组织内主要成分为胶原，可以进行后续生物墨水制备。
由图5可知，添加了高分子材料的生物墨水的动态弹性模量均大于未添加的生物墨水；可见，在生物墨水中添加高分子材料，能够显著提高其力学性能，并以此制得性能更好的仿生人工颞下颌关节盘。
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做出的各种修改和变形仍属本专利的保护范围。

Claims (7)

1.一种仿生人工颞下颌关节盘的制备方法，其特征在于，包括以下步骤：
（1）在无菌条件下收取牛尾的椎间盘，清洗，然后在-20~37℃温度区间冻融处理4~6个循环，采用超纯水/盐酸胍化学试剂提取糖胺聚糖和蛋白，再将剩余组织打碎至絮状，加入4~6wt%的十二烷基硫酸钠和4~6 KU/ml的DNA酶室温处理2~4h，无酶水洗净，得椎间盘絮状样品；
（2）将步骤（1）所得椎间盘絮状样品冷冻干燥，然后加入消化液，在室温、25~35 r/min条件下共混60~84h，调节pH值至中性，再加入步骤（1）所得糖胺聚糖和蛋白，得生物墨水；所述消化液通过以下方法制备得到：将胃蛋白酶溶于0.4~0.6vt%的醋酸溶液中，得消化液；所述胃蛋白酶与所述椎间盘絮状样品干重质量比为1:9~11；
（3）将生物相容性好的高分子材料和步骤（2）所得生物墨水按质量比1:1~3在室温下共混，然后进行3D打印，得仿生人工颞下颌关节盘；所述生物相容性好的高分子材料为降冰片烯改性透明质酸或甲基丙烯酸化水凝胶。
2.如权利要求1所述的仿生人工颞下颌关节盘的制备方法，其特征在于，步骤（1）中，在无菌条件下收取8月龄牛尾的椎间盘，用生理盐水清洗去除血迹。
3.如权利要求1所述的仿生人工颞下颌关节盘的制备方法，其特征在于，步骤（1）中，加入5wt%的十二烷基硫酸钠和5 KU/ml的DNA酶室温处理3h。
4.如权利要求1所述的仿生人工颞下颌关节盘的制备方法，其特征在于，所述醋酸溶液浓度为0.5vt%，所述胃蛋白酶与所述椎间盘絮状样品干重质量比为1:10。
5.如权利要求1所述的仿生人工颞下颌关节盘的制备方法，其特征在于，步骤（2）中，用氢氧化钠调节pH值至中性。
6.如权利要求1所述的仿生人工颞下颌关节盘的制备方法，其特征在于，步骤（3）中，高分子材料和生物墨水按质量比1:1在室温下共混。
7.权利要求1~6任一项所述的仿生人工颞下颌关节盘的制备方法制得的仿生人工颞下颌关节盘。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105030828A (zh) * 2008-06-25 2015-11-11 麦瑟布莱斯特公司 椎间盘的修复和/或重建
CN106474541A (zh) * 2015-08-31 2017-03-08 华东理工大学 一种3d的组织工程支架及其制备方法
WO2017044570A1 (en) * 2015-09-08 2017-03-16 Clemson University Decellularized biomaterial and method for formation
CN106562842A (zh) * 2016-11-09 2017-04-19 广州医科大学附属口腔医院 一种基于三维打印的人工颞下颌关节

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103750923A (zh) * 2013-12-20 2014-04-30 中山大学附属口腔医院 基于选择性激光熔化技术的人工颞下颌关节及其制造方法
US11052175B2 (en) * 2015-08-19 2021-07-06 Musculoskeletal Transplant Foundation Cartilage-derived implants and methods of making and using same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105030828A (zh) * 2008-06-25 2015-11-11 麦瑟布莱斯特公司 椎间盘的修复和/或重建
CN106474541A (zh) * 2015-08-31 2017-03-08 华东理工大学 一种3d的组织工程支架及其制备方法
WO2017044570A1 (en) * 2015-09-08 2017-03-16 Clemson University Decellularized biomaterial and method for formation
CN106562842A (zh) * 2016-11-09 2017-04-19 广州医科大学附属口腔医院 一种基于三维打印的人工颞下颌关节

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