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

Info

Publication number
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
Authority
CN
China
Prior art keywords
cartilage
preparation
concentration
cell
protection liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610297085.3A
Other languages
English (en)
Other versions
CN105879118A (zh
Inventor
史真
史伟云
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baiodisel (Chengdu) Biotechnology Co., Ltd
Original Assignee
Diesel (beijing) Biological Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Diesel (beijing) Biological Technology Co Ltd filed Critical Diesel (beijing) Biological Technology Co Ltd
Priority to CN201610297085.3A priority Critical patent/CN105879118B/zh
Publication of CN105879118A publication Critical patent/CN105879118A/zh
Application granted granted Critical
Publication of CN105879118B publication Critical patent/CN105879118B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Molecular Biology (AREA)
  • Vascular Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Materials For Medical Uses (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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。
CN201610297085.3A 2016-05-09 2016-05-09 一种生物工程脱细胞软骨的制备方法 Active CN105879118B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610297085.3A CN105879118B (zh) 2016-05-09 2016-05-09 一种生物工程脱细胞软骨的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610297085.3A CN105879118B (zh) 2016-05-09 2016-05-09 一种生物工程脱细胞软骨的制备方法

Publications (2)

Publication Number Publication Date
CN105879118A CN105879118A (zh) 2016-08-24
CN105879118B true CN105879118B (zh) 2018-03-02

Family

ID=56703373

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610297085.3A Active CN105879118B (zh) 2016-05-09 2016-05-09 一种生物工程脱细胞软骨的制备方法

Country Status (1)

Country Link
CN (1) CN105879118B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721698A (zh) * 2017-04-24 2018-11-02 上海市肺科医院 一种组织工程软骨支架及其制备方法和用途
CN109550081B (zh) * 2018-11-27 2021-07-16 中国人民解放军总医院第四医学中心 一种脱抗原神经处理方法
CN113577389A (zh) * 2021-08-06 2021-11-02 中国医学科学院整形外科医院 一种来自猪耳软骨的脱细胞软骨材料及制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102470149A (zh) * 2009-08-18 2012-05-23 生命细胞公司 用于处理组织的方法
CN102573945A (zh) * 2009-09-11 2012-07-11 翰林大学校产学协力团 冷冻保存无细胞真皮基质的制备方法及由其制备的冷冻保存无细胞真皮基质
CN104971380A (zh) * 2014-04-11 2015-10-14 烟台隽秀生物科技有限公司 一种脱细胞基质修复凝胶及其制备新方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6734018B2 (en) * 1999-06-07 2004-05-11 Lifenet Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants produced

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102470149A (zh) * 2009-08-18 2012-05-23 生命细胞公司 用于处理组织的方法
CN102573945A (zh) * 2009-09-11 2012-07-11 翰林大学校产学协力团 冷冻保存无细胞真皮基质的制备方法及由其制备的冷冻保存无细胞真皮基质
CN104971380A (zh) * 2014-04-11 2015-10-14 烟台隽秀生物科技有限公司 一种脱细胞基质修复凝胶及其制备新方法

Also Published As

Publication number Publication date
CN105879118A (zh) 2016-08-24

Similar Documents

Publication Publication Date Title
US11957814B2 (en) Adipose tissue matrices
CN103432627B (zh) 一种制备动物脱细胞组织基质材料的方法及其制备的组织基质材料
CN101384704B (zh) 可再吸收的角膜扣状体
JP6889118B2 (ja) 高純度コラーゲン粒子の製造及びその使用
CN102580153A (zh) 一种制备脱细胞真皮基质医用组织补片的方法
CN105879118B (zh) 一种生物工程脱细胞软骨的制备方法
CN103785065B (zh) 一种软骨再生支架材料的制备方法
CN104055795B (zh) 一种可注射植入剂及其制备方法
CN102327640A (zh) 一种脱细胞羊膜的制备方法
CN101773687A (zh) 一种复合软组织补片的制备方法
CN105944142B (zh) 一种脱细胞肌腱或韧带支架的制备方法
WO2011118954A3 (ko) 사람 하비갑개 유래 중간엽 기질세포로부터 연골, 골, 신경세포 또는 지방세포를 분화시키는 방법
CN107058217A (zh) 一种软骨细胞的增殖培养基及增殖培养方法
CN105833353B (zh) 一种生物工程脱细胞真皮基质的制备及用途
CN101695583B (zh) 一种用于组织修复的颗粒状生物材料及其制备方法
WO2004011631A3 (en) Methods and compositions for treating tissue defects using pulsed electromagnetic field stimulus
CN102580152B (zh) 一种制备脱细胞骨的方法
CN103418001A (zh) 一种动物组织材料的消毒灭菌方法以及相应的动物组织浸泡溶液
CN102921034A (zh) 猪脱细胞真皮基质包被胶原的复合敷料及其制备方法
EP3188596B1 (en) Human dermis, preparation and use thereof
CN104548205B (zh) 一种动物源神经支架及其制备方法
CN102580140B (zh) 一种制备脱细胞生物膜敷料的方法
CN105833356B (zh) 一种新型的脱细胞带瓣血管支架及其制备方法
CN109771697B (zh) 一种真皮成纤维细胞皮片及其构建方法和应用
Mumme Cartilage tissue engineering from Nose 2Kne: 12-months results of a phase 1 clinical trial

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20211112

Address after: 611139 floors 1-4, building 15, No. 1919, Shuangyan Road, cross strait science and Technology Industrial Development Park, Wenjiang District, Chengdu, Sichuan

Patentee after: Baiodisel (Chengdu) Biotechnology Co., Ltd

Address before: 100176 601a, 6 / F, building 13, yard 8, Liangshuihe Second Street, Beijing Economic and Technological Development Zone, Beijing

Patentee before: Baiodisel (Beijing) Biotechnology Co., Ltd