CN105944142B - 一种脱细胞肌腱或韧带支架的制备方法 - Google Patents

一种脱细胞肌腱或韧带支架的制备方法 Download PDF

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CN105944142B
CN105944142B CN201610297084.9A CN201610297084A CN105944142B CN 105944142 B CN105944142 B CN 105944142B CN 201610297084 A CN201610297084 A CN 201610297084A CN 105944142 B CN105944142 B CN 105944142B
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史真
史伟云
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Baiodisel (Chengdu) Biotechnology Co.,Ltd.
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Abstract

本发明涉及一种脱细胞肌腱或韧带支架的制备方法。目的在于提供一种具备良好的生物力学特性和生物相容性的脱细胞肌腱或韧带胶原纤维材料。获取去除外膜和滑膜组织的动物肌腱或韧带，脱细胞过程中全程使用含有韧带或肌腱支架结构保护剂的混合保护液；采用高静压技术预分离韧带或肌腱组织内的细胞；采用复合核酸酶方法消化残余细胞核；利用去垢剂脱除松散破碎细胞片；使用混合保护液进行漂洗。本发明通过高效的物理，少量的酶和去垢剂脱的方法充分去除韧带或肌腱中的细胞成分，并应用保护液的全程保护，以保证细胞核脱净的基础上，最大限度地降低肌腱或韧带组织的免疫原性的同时保留了其正常胶原纤维的三维结构及排列极性。

Description

一种脱细胞肌腱或韧带支架的制备方法
技术领域
本发明涉及生物工程医用材料的制备领域，具体为脱细胞肌腱/韧带支架的制备方法。
背景技术
韧带和肌腱损伤后修复后愈合能力差是临床上常见的难题，使用替代物应用于韧带和肌腱的修复重建是临床急需的。目前肌腱和韧带替代物主要包括自体移植物、异体或异种移植物及合成材料移植物，但均有其不可回避的缺点。虽然自体韧带移植为最有效的修复重建方法，但限于取材的范围和量，不能广泛应用于临床。异体或异种肌腱抗原性强，术后免疫排斥不可避免是其最大的缺点。人工合成材料如碳纤维、聚酯材料等非降解材料，长期使用易引起磨损断裂。理想的生物替代材料应该具备良好的生物力学特性和生物相容性。肌腱/韧带脱细胞胶原纤维材料为一种新型的生物材料，保留有肌腱/韧带固有的力学结构单位和细胞附着结构，去除了细胞成分，具有良好的力学性能和生物相容性。随着脱细胞技术及灭菌技术的发展，宿主对异体、异种韧带支架的免疫排斥和疾病传染已大大降低，使得韧带和肌腱脱细胞支架在临床应用成为可能。因韧带和肌腱是致密的结缔组织，纤维束内外有内膜和外膜包绕使得细胞成分脱除困难，所以脱细胞周期长，脱细胞过程对细胞外基质破坏较大。目前在制备用于体内修复的肌腱/韧带时，所使用的方法很难在既有效脱细胞、去异种抗原的同时，又保留肌腱/韧带组织的结构形态、生物力学特性和生物特性。因此急需一种稳定高效的脱细胞方法来克服此难题。
发明内容
本发明提供了一种快速、全程使用特殊保护液以及高效的物理复合生物酶手段制备肌腱/韧带脱细胞支架的方法。
本发明的创新处在于：1、肌腱/韧带脱细胞过程中全程使用包含一种或多种肌腱/韧带结构保护成分的混合保护液；2、采用高静压技术预分离肌腱/韧带组织内的细胞；3、采用复合核酸酶方法消化残余细胞核；4、利用去垢剂脱除松散破碎细胞片；5、使用混合保护液进行漂洗。脱细胞步骤设定合适的温度、时间以及去垢剂、消化酶浓度对肌腱/韧带组织进行处理。
各种保护性成分的用量为硫酸软骨素的浓度为20-60g/L；透明质酸的浓度为10-50g/L；右旋糖酐的浓度为5-20g/L；红霉素5-8.5mg/L。
高静压压力条件为500-800MPa，高静压频率为3～8次，每次为5～8分钟。
DNA酶的浓度为2500-5000U/ml，核酸酶的浓度为2500-5000U/ml。DNA酶或核酸酶的脱细胞核时间为4-8小时，处理温度为15-30度。
去垢剂条件为十二烷基磺酸钠的浓度0.1-5％，海藻酸钠的浓度0.1-5％，TritonX-100％的浓度5-10％。去垢剂处理时间为4-8小时。
可选择性的同时或分开进行酶和去垢剂的处理。
制备的肌腱/韧带材料需贴附在硝酸甘油膜上，放入无水氯化钙分子筛内脱水，经装袋密封并标记，经钴60照射后在4℃下保存备用。
具体实施方式
下面通过实施例对本发明进行具体的描述，这些实施例只用于进一步详述说明本发明，不能理解为对本发明保护范围的限定，在本发明保护范围之内做出非本质的调整需本发明方授权。
本发明的实施例1
取新鲜(宰后3小时内取得)猪或牛大腿肌腱或韧带组织，去除表面的外膜和滑膜组织后，留取1X2cm的肌腱/韧带组织片备用。
1、全程使用保护液对肌腱/韧带组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素20g/L，右旋糖酐15g/L，红霉素5mg/L，调pH值至7.2，渗透压为400mOsm；
2、将肌腱/韧带组织片密封在盛有保护液的塑料袋中；
3、于500MPa高静压条件下，处理8次，每次时间为5分钟；
4、将肌腱/韧带取出后，置于含5％Triton X-100+2500U/ml DNA酶的保护液中，温度为30℃，设定摇床速度为100转/分钟，处理5小时；
5、去垢剂与酶处理完毕后，取出肌腱/韧带置于保护液中漂洗4小时。
本发明的实施例2
取新鲜(宰后3小时内取得)猪或牛大腿肌腱或韧带组织，去除表面的外膜和滑膜组织后，留取1X2cm的肌腱/韧带组织片备用。
1、全程使用保护液对肌腱/韧带组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素50g/L，透明质酸20g/L，红霉素6mg/L，调pH值至7.2，渗透压为450mOsm；
2、将肌腱/韧带组织片密封在盛有保护液的塑料袋中；
3、于600MPa高静压条件下，处理6次，每次时间为6分钟；
4、将肌腱/韧带取出后，置于含5％十二烷基磺酸钠+4000U/ml DNA酶的保护液中，温度为25℃，设定摇床速度为100转/分钟，处理5小时；
5、去垢剂与酶处理完毕后，取出肌腱/韧带置于保护液中漂洗6小时。
本发明的实施例3
取新鲜(宰后3小时内取得)猪或牛大腿肌腱或韧带组织，去除表面的外膜和滑膜组织后，留取1X2cm的肌腱/韧带组织片备用。
1、全程使用保护液对肌腱/韧带组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素30g/L，透明质酸50g/L，新霉素5mg/L，调pH值至7.2，渗透压为450mOsm；
2、将肌腱/韧带组织片密封在盛有保护液的塑料袋中；
3、于800MPa高静压条件下，处理4次，每次时间为5分钟；
4、将肌腱/韧带取出后，置于含2％海藻酸钠+5000U/ml DNA酶的保护液中，温度为30℃，设定摇床速度为100转/分钟，处理8小时；
5、去垢剂与酶处理完毕后，取出肌腱/韧带置于保护液中漂洗5小时。

Claims (9)

1.一种脱细胞肌腱或韧带支架材料的制备方法，其特征在于包括以下步骤：
(1)取动物肌腱或韧带，清除其表面的外膜和滑膜组织；
(2)全程使用包含一种或多种保护肌腱或韧带功能和组织完整性的混合保护液；
(3)使用高静压预分离肌腱或韧带内的细胞；
(4)采用复合核酸酶去除破碎细胞核；
(5)利用去垢剂脱除松散破碎细胞；
(6)在肌腱或韧带混合保护液中漂洗去除多余的核酸酶和细胞碎片；
(7)获得具有正常胶原纤维的三维结构及排列极性的带有生物特性的脱细胞肌腱或韧带；
其中所述混合保护液成分组成为：RPMI-1640培养基；硫酸软骨素的浓度为20-60g/L；透明质酸的浓度为10-50g/L；右旋糖酐的浓度为10-20g/L；妥布霉素5-8.5mg/L，调节pH值至6.8-7.2之间，渗透压为400-500mOsm；
所述高静压压力条件为500-800MPa，高静压频率为3-8次，每次为5～8分钟。
2.如权利要求1所述的制备方法，取宽度为1-5cm的成年猪或牛大腿部的肌腱或韧带备用。
3.如权利要求1所述的制备方法，其中所述复合核酸酶包括DNA酶、RNA酶、其他核酸酶及其组合。
4.如权利要求1所述的制备方法，其中所述去垢剂包括Triton、十二烷基磺酸钠、十二烷基肌氨酸钠、海藻酸钠、CHAPS活性剂中的一种或多种作为组合。
5.如权利要求1所述的制备方法，其中在肌腱/韧带混合保护液中漂洗去除多余的核酸酶和细胞碎片中漂洗的时间为4～8小时。
6.如权利要求1所述的制备方法，制备的肌腱或韧带支架材料需贴附在硝酸甘油模上，放入无水氯化钙分子筛内脱水，经装袋密封并标记，经钴60照射后在4℃下保存备用。
7.如权利要求3所述的制备方法，其中所述复合核酸酶使用DNA酶时DNA酶的浓度为2500-5000U/mL，DNA酶的脱细胞核时间为4-8小时，处理温度为15-30摄氏度。
8.如权利要求3所述的制备方法，其中所述复合核酸酶使用核酸酶时浓度为2500-5000U/mL，核酸酶的脱细胞核时间为4-8小时，处理温度为15-30摄氏度。
9.如权利要求4所述的制备方法，其中所述去垢剂十二烷基磺酸钠的浓度为0.1-5％，十二海藻酸钠的浓度为0.1-5％，Triton X-100的浓度为5-10％，去垢剂使用时间为4-8小时。
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