CN105879118B - 一种生物工程脱细胞软骨的制备方法 - Google Patents

一种生物工程脱细胞软骨的制备方法 Download PDF

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CN105879118B
CN105879118B CN201610297085.3A CN201610297085A CN105879118B CN 105879118 B CN105879118 B CN 105879118B CN 201610297085 A CN201610297085 A CN 201610297085A CN 105879118 B CN105879118 B CN 105879118B
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史真
史伟云
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Baiodisel (Chengdu) Biotechnology Co., Ltd
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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
    • A61L27/3612—Cartilage, synovial fluid
    • 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
    • A61L27/3654—Cartilage, e.g. meniscus
    • 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
    • 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
    • A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Abstract

本发明涉及一种新型的生物工程脱细胞软骨及其制备方法。对切为片状或特定的形状的动物软骨进行脱细胞，脱细胞过程中全程使用含有软骨结构保护剂的混合保护液；采用高静压技术预分离软骨组织内的细胞；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软骨中的细胞成分，并应用保护液的全程保护，以保证细胞核脱干净的基础上，最大限度地降低软骨中的免疫原性，同时保留了软骨的正常胶原纤维的三维结构及排列极性，使制得的脱细胞软骨具有良好的生物相容性、生物力学性与骨诱导能力。

Description

一种生物工程脱细胞软骨的制备方法
技术领域
本发明涉及生物工程医用材料的制备领域，具体为脱细胞软骨的制备方法。
背景技术
因机械创伤和烧伤，自身免疫相关性炎症、退变形疾病和肿瘤切除等原因导致的某些部位皮肤和软骨的缺损，关节软骨损伤是临床上常见的疾病。如眼睑的外伤和肿瘤，在切除病变的眼睑后，眼球就会暴露，导致角膜溃疡最终眼球难保，临床修复时最大的困难不是眼睑的皮肤，而是需要理想的睑板支撑物。再如，关节软骨再生及修复能力极为有限，一旦损伤后，难以自身修复。目前的治疗方法，自体或异体软骨膜移植，都存在着许多缺陷，从而限制了临床应用。随着生物组织工程技术的出现，应用生物工程技术制备工程软骨或骨软骨复合体被认为解决这一问题最好的技术手段。
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软骨脱细胞材料的方法。
本发明的发明要点在于：1、软骨脱细胞过程中全程使用包含一种或多种软骨结构保护成分的混合保护液；2、采用高静压技术预分离软骨组织内的细胞；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、消化酶浓度对软骨组织进行处理。
各种保护性成分的用量为硫酸软骨素的浓度为50-100g/L；透明质酸的浓度为20-60g/L；右旋糖酐的浓度为15-30g/L；链霉素5-10mg/L。
高静压压力条件为600-1000MPa，高静压频率为5～10次，每次为6～8分钟。
DNA酶的浓度为3000-6000U/ml，核酸酶的浓度为3000-6000U/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硫酸钠的浓度0.1-5％，十二烷基肌氨酸钠的浓度0.1-5％，CHAPS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。
制备的软骨材料放入无水氯化钙分子筛内脱水，经装袋密封并标记，用辐照剂量为25kGy的Gamma射线辐照灭菌5-10小时后在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
取新鲜成年猪耳廓软骨，切成1X2cm大小，厚度0.1-0.30mm的薄片备用。
1、全程使用保护液对软骨组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素50g/L，右旋糖酐15g/L，链霉素5mg/L，调pH值至7.2，渗透压为450mOsm；
2、将软骨组织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10次，每次时间为6分钟；
4、将软骨取出后，置于含5％CHAPS+3000U/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，取出软骨置于保护液中漂洗8小时。
本发明的实施例2
取新鲜成年猪耳廓软骨，切成1X2cm大小，厚度0.1-0.30mm的薄片备用。
1、全程使用保护液对软骨组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素80g/L，透明质酸50g/L，链霉素5mg/L，调pH值至7.2，渗透压为500mOsm；
2、将软骨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8次，每次时间为6分钟；
4、将软骨取出后，置于含5％十二烷基硫酸钠+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，取出软骨置于保护液中漂洗6小时。
本发明的实施例3
取新鲜成年猪耳廓软骨，切成1X2cm大小，厚度0.1-0.30mm的薄片备用。
1、全程使用保护液对软骨组织进行保护，保护液配方为：RPMI-1640培养基，添加硫酸软骨素80g/L，透明质酸50g/L，链霉素5mg/L，调pH值至7.2，渗透压为550mOsm；
2、将软骨片密封在盛有保护液的塑料袋中；
3、于1000MPa高静压条件下，处理5次，每次时间为6分钟；
4、将软骨取出后，置于含2％十二烷基肌氨酸钠+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，取出软骨置于保护液中漂洗5小时。

Claims (10)

1.一种生物工程脱细胞软骨的制备方法，其特征在于包括以下步骤：
(1)取动物软骨；
(2)使用包含一种或多种保护软骨功能和组织完整性的混合保护液；
(3)使用高静压预分离软骨内的细胞；
(3)采用复合核酸酶去除破碎细胞核；
(4)利用去垢剂脱除松散破碎细胞；
(5)在软骨混合保护液中漂洗去除多余的核酸酶和细胞碎片；
(6)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软骨；
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50-100g/L；透明质酸的浓度为20-60g/L；右旋糖酐的浓度为15-30g/L；链霉素5-10mg/L，调节pH值为6.8-7.2，渗透压为450-550mOsm。
2.如权利要求1所述的制备方法，所取软骨的厚度在0.2-1.0mm之间，长宽为0.5cm和1cm的片状或特定形状，所述软骨来自猪耳朵或牛耳朵或关节软骨。
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十二烷基硫酸钠、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，其中在软骨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5～10小时。
6.如权利要求1所述的制备方法，制备的软骨材料放入无水氯化钙分子筛内脱水，经装袋密封并标记，用辐照剂量为25kGy的Gamma射线辐照灭菌5-10小时后在4℃下保存备用。
7.如权利要求1所述的制备方法，其中所述高静压的压力条件为600-1000MPa，所述高静压的频率为5-10次，每次6～8分钟。
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3000-6000U/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3所述的制备方法，其中所述复合核酸酶使用DNA酶时浓度为3000-6000U/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。
10.如权利要求4所述的制备方法，其中所述去垢剂十二烷基硫酸钠的浓度为0.1-5％，十二烷基肌氨酸钠的浓度为0.1-5％，CHAPS的浓度为5-10％，去垢剂使用时间为4-8小时。
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