CN109675113A - 一种组织工程肌腱微组织的制备方法 - Google Patents

一种组织工程肌腱微组织的制备方法 Download PDF

Info

Publication number
CN109675113A
CN109675113A CN201811470046.4A CN201811470046A CN109675113A CN 109675113 A CN109675113 A CN 109675113A CN 201811470046 A CN201811470046 A CN 201811470046A CN 109675113 A CN109675113 A CN 109675113A
Authority
CN
China
Prior art keywords
tendon
cell
tenocyte
assembly robot
micro
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.)
Pending
Application number
CN201811470046.4A
Other languages
English (en)
Inventor
彭江
亓建洪
张凯红
孟昊业
汪爱媛
汤贺
王玉
许文静
孙百川
刘雪剑
柳昊
袁光华
卢世璧
郭全义
刘舒云
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.)
Chinese PLA General Hospital
Taishan Medical University
Original Assignee
Chinese PLA General Hospital
Taishan Medical University
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 Chinese PLA General Hospital, Taishan Medical University filed Critical Chinese PLA General Hospital
Priority to CN201811470046.4A priority Critical patent/CN109675113A/zh
Publication of CN109675113A publication Critical patent/CN109675113A/zh
Pending legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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 containing added animal cells
    • A61L27/3804Materials 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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/3604Materials 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
    • A61L27/3633Extracellular matrix [ECM]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/3641Materials 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
    • A61L27/3645Connective tissue
    • A61L27/3662Ligaments, tendons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/3683Materials 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
    • A61L27/3687Materials 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/3683Materials 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
    • A61L27/3691Materials 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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 containing added animal cells
    • A61L27/3839Materials 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 containing added animal cells characterised by the site of application in the body
    • A61L27/3843Connective tissue
    • A61L27/386Ligaments, tendons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412Tissue-regenerating or healing or proliferative agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/10Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/40Preparation 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)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Vascular Medicine (AREA)
  • Rheumatology (AREA)
  • Urology & Nephrology (AREA)
  • Rehabilitation Therapy (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开一种组织工程肌腱微组织的制备方法,属于生物医药技术领域,旨在构建一种生物源性材料,可在体外与干细胞构建肌腱微组织诱导其向肌腱方向分化,促进肌腱特定蛋白Tenomodulin以及一型和三型胶原的表达,为修复肌腱损伤提供高品质生物材料。该方法的步骤为:将大鼠尾腱取出,反复冻融若干次,在摇床上经过SDS溶液于室温下处理,制备成脱细胞的肌腱细胞外基质;将上述脱细胞的肌腱细胞外基质低温定型,经真空冷冻干燥,获得成型的肌腱细胞外基质;将上述成型的肌腱细胞外基质与P2代大鼠脂肪干细胞共培养若干天,制备出组织工程肌腱微组织。

Description

一种组织工程肌腱微组织的制备方法
技术领域
本发明属于生物医药技术领域,尤其涉及一种组织工程肌腱微组织的制备方法。
背景技术
肌腱是维持关节稳定以及正常运动的重要结缔组织,由于肌腱组织细胞分布和营养血供较少,一旦损伤其自我修复能力较差。如骨性关节炎等,严重甚至影响关节的正常功能。肌腱损伤是骨科的一种常见病,每年在全球范围内有超过3000万肌腱和韧带损伤病例发生,其中大多数与体育相关;然而,用目前的治疗手段很难使损伤的肌腱完全恢复到伤前的状态。因此,如何提高损伤肌腱的愈合效果以及最大限度恢复其正常功能是目前医学面临的一项重大难题。
近些年来,组织工程技术逐渐运用在肌腱修复的领域,其中最关键的部分是选择合适的支架。理想的组织工程支架应该具备两点特性:(1)有较好的生物相容性,能够为种子细胞的生长分化提供一个合适的环境;(2)具备良好的生物力学特性。动物实验和临床研究显示,脱细胞肌腱能够降低组织的免疫源性,使其与宿主以及种子细胞产生良好的生物相容性,用于修复肌腱损伤能在一定程度上恢复肌腱正常功能。此外,细胞外基质(ECM)能为种子细胞提供模拟正常机体的生长微环境,对维持干细胞特性有重要作用。
细胞外基质内含各种细胞因子,它们对跟腱正常生理功能以及病理的愈合起着至关重要的调控作用。在正常组织中,细胞外基质是一个复杂的网状大分子结构,占据着细胞间的空间,主要负责细胞间的信号传导、生长增殖的调控以及力学支撑。据文献报道,肌腱细胞外基质富含大量的基质蛋白,它能促进种子细胞增殖以及向肌腱方向分化,同时维持干细胞表型抑制异位骨化。此外,肌腱细胞外基质源性支架明显增强修复组织的力学性能,同时通过抑制或者激活种子细胞的基质金属蛋白酶(MMPs)来调控肌腱损伤的修复过程。
脂肪间充质干细胞(ADSCs)作为肌腱组织工程的种子细胞,是目前研究最多也是使用最多的种子细胞之一。ADSCs有取材简单,使用方便,对机体损伤小等优点,而且可以通过抽取少量脂肪而获得,它在体外培养过程中可以保持未分化表型在短时间内大量增殖。有研究证明在体外培养过程中,ADSCs未经诱导即具备分泌I型胶原的能力,这与肌腱细胞的功能相像。有研究检测出在普通培养状态下的ADSCs即具备分泌I型胶原的能力,并且在接种后透射电镜显示胞体内含有丰富的粗面内质网,说明在共培养条件下ADSCs仍保持旺盛的分泌功能。因此,ADSCs是肌腱组织工程中一种理想的种子细胞来源。尽管有部分研究者证明可以在体外诱导ADSCs定向分化为肌腱细胞,但是到目前为止,体外培养条件下使ADSCs定向分化为肌腱细胞的技术手段并不成熟。值得注意的是在体外培养的ADSCs移植入体内后,有向肌腱组织分化的倾向,这可能与受体局部微环境中相关的细胞因子的促进与诱导有关。由此可见在肌腱组织工程中,种子细胞的微环境对其生长分化起着决定性的作用。因此我们将脂肪间充质干细胞复合肌腱细胞外基质制备组织工程肌腱微组织,使种子细胞处于模拟肌腱组织的微环境中,促进种子细胞向肌腱方向生长分化,希望能够提高肌腱损伤的修复效果。
发明内容
本发明的目的在于提供一种组织工程肌腱微组织的制备方法,旨在构建一种生物源性材料,可在体外与干细胞构建肌腱微组织诱导其向肌腱方向分化,促进肌腱特定蛋白Tenomodulin以及一型和三型胶原的表达,为修复肌腱损伤提供高品质生物材料。
为实现上述目的,本发明采用如下技术方案:
一种组织工程肌腱微组织的制备方法,步骤包括:
将大鼠尾腱取出,反复冻融若干次,在摇床上经过SDS(十二烷基硫酸钠)溶液于室温下处理,制备成脱细胞的肌腱细胞外基质;
将上述脱细胞的肌腱细胞外基质低温定型,经真空冷冻干燥,获得成型的肌腱细胞外基质;
将上述成型的肌腱细胞外基质与P2代大鼠脂肪干细胞共培养若干天,制备出组织工程肌腱微组织。
进一步地,所述大鼠尾腱取出的方法为:取若干只健康的150-200g重的SD大鼠鼠尾,按关节段分离成小段,再在无菌条件下取出大鼠尾腱,经PBS溶液彻底清洗后置于含1%~5%双氧水的容器中,0~25℃下过夜。
进一步地,充分洗净所述大鼠尾腱上的多余双氧水,加入PBS溶液淹没尾腱,并将其反复冻融3~5次。
进一步地,所述SDS溶液浓度为1%~3%,处理时间为12~48h。
进一步地,对所述脱细胞的肌腱细胞外基质采用病理染色、抗原检测的方法评价其脱细胞的效果。
进一步地,将所述脱细胞的肌腱细胞外基质先后置于-10~-40℃冰箱、-60~-110℃冰箱中以低温定型。
进一步地,将所述成型的肌腱细胞外基质用密封袋贮存密封,再用钴60消毒,4℃下保存备用。
进一步地,所述培养天数为7~14天。
一种利用上述方法制备的组织工程肌腱微组织。
与现有技术相比,本发明的方法能够有效去除尾腱细胞,大大降低肌腱组织的抗原性,与脂肪间充质干细胞有较好的生物相容性,在共培养一定天数后,脂肪间充质干细胞大量扩增,细胞活性增强。本发明所制备的组织工程肌腱微组织为生物来源性材料,真空冷冻干燥后有一定的生物力学性能,符合理想支架的最基本要求。使用本发明制备的组织工程肌腱微组织作为材料用于大鼠和比格犬跟腱缺损修复,在术后实验组的跟腱修复质量(大体观察、胶原和TNMD表达、组织染色、影像学观察、力学检测等)明显提高,显示其用于肌腱组织损伤修复的良好效果和应用前景。
附图说明
图1是实施例中提供的组织工程肌腱微组织制备方法流程图。
图2A-2D是脱细胞前后的组织学染色图;其中图2A、2B为脱细胞前后HE染色图,图2C、2D为脱细胞前后DAPI染色图。
图3是脱细胞前后组织α-Gal抗原含量的检测数据图。
图4是脱细胞后组织生物相容性检测数据图。
图5A-5B是肌腱微组织的构建图,其中图5A为培养1天时的图,图5B为培养5天时的图。
具体实施方式
为使本发明的上述特征和优点能更明显易懂,下文特举实施例,并配合所附图作详细说明如下。
本实施例提供一种组织工程肌腱微组织的制备方法,如图1所示,该方法包括以下步骤:
S101:将20只健康的150-200g重的SD大鼠的尾巴按照关节切成小段,充分取出大鼠尾腱;
S102:大鼠尾腱取出反复冻融3次,经过经过1%SDS溶液于室温下处理24h;
S103:尾腱脱细胞处理后先后置于-20℃冰箱、-80℃低温冰箱使其定型,真空冷冻干燥获得成型的肌腱细胞外基质,密封袋内贮存密封,钴60消毒,4℃保存备用;
S104:肌腱细胞外基质与P2代大鼠脂肪干细胞共培养7天,制备出组织工程肌腱微组。
采用本发明的方法制备组织工程肌腱微组织,通过反复冻融结合1%SDS溶液处理的脱细胞方法能有效的去除组织中的细胞,如图2A-2D所示,其中图2A、2B为脱细胞前后HE染色图,图2C、2D为脱细胞前后DAPI染色图。
通过图3可知,脱细胞后尾腱组织的α-Gal含量显著低于脱细胞前,该差异具有统计学意义,表明极大地降低了组织的免疫源性。
通过图4所反映的检索数据来看,在培养周期内的各时间点,实验组与以单纯DMEM培养液为对照组的OD值差异无统计学意义,即实验组与单纯DMEM对于细胞的生长、增殖的影响无差异,实验组的该种肌腱细胞外基质具有良好的生物相容性,说明制备的组织工程肌腱微组织与脂肪间充质干细胞有较好的生物相容性。
图5A-5B为肌腱微组织在培养过程中的构建图,其中图5A显示:培养1天时大量活细胞贴附,并且有较高的活性,少量死细胞出现;图5B显示:随着培养的时间延长,脂肪间充质干细胞持续扩增,到7天时细胞活性也明显提高;由此可见,在共培养7天后脂肪间充质干细胞大量扩增,细胞活性增强。
以上实施例仅列举了本发明内容公开的参数的其中一种情况,仅用以说明本发明的技术方案,而非对其进行限制,在其他的实施例中,也可以选择如下参数范围“1%~5%双氧水、过夜温度0~25℃、反复冻融3~5次、SDS溶液浓度1%~3%、处理时间12~48h、脱细胞的肌腱细胞外基质先后置于-10~-40℃冰箱、-60~-110℃冰箱中、培养天数为7~14天”中的任意一种情况,本领域的普通技术人员可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明的精神和范围,本发明的保护范围应以权利要求书所述为准。

Claims (9)

1.一种组织工程肌腱微组织的制备方法,步骤包括:
将大鼠尾腱取出,反复冻融若干次,在摇床上经过SDS溶液于室温下处理,制备成脱细胞的肌腱细胞外基质;
将上述脱细胞的肌腱细胞外基质低温定型,经真空冷冻干燥,获得成型的肌腱细胞外基质;
将上述成型的肌腱细胞外基质与P2代大鼠脂肪干细胞共培养若干天,制备出组织工程肌腱微组织。
2.如权利要求1所述的方法,其特征在于,所述大鼠尾腱取出的方法为:取若干只健康的150-200g重的SD大鼠鼠尾,按关节段分离成小段,再在无菌条件下取出大鼠尾腱,经PBS溶液彻底清洗后置于含1%~5%双氧水的容器中,0~25℃下过夜。
3.如权利要求2所述的方法,其特征在于,充分洗净所述大鼠尾腱上的多余双氧水,加入PBS溶液淹没尾腱,并将其反复冻融3~5次。
4.如权利要求1所述的方法,其特征在于,所述SDS溶液浓度为1%~3%,处理时间为12~48h。
5.如权利要求1所述的方法,其特征在于,对所述脱细胞的肌腱细胞外基质采用病理染色、抗原检测的方法评价其脱细胞的效果。
6.如权利要求1所述的方法,其特征在于,将所述脱细胞的肌腱细胞外基质先后置于-10~-40℃冰箱、-60~-110℃冰箱中以低温定型。
7.如权利要求1所述的方法,其特征在于,将所述成型的肌腱细胞外基质用密封袋贮存密封,再用钴60消毒,4℃下保存备用。
8.如权利要求1所述的方法,其特征在于,所述培养天数为7~14天。
9.一种利用上述权利要求1~8任一所述的方法制备的组织工程肌腱微组织。
CN201811470046.4A 2018-11-26 2018-11-26 一种组织工程肌腱微组织的制备方法 Pending CN109675113A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811470046.4A CN109675113A (zh) 2018-11-26 2018-11-26 一种组织工程肌腱微组织的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811470046.4A CN109675113A (zh) 2018-11-26 2018-11-26 一种组织工程肌腱微组织的制备方法

Publications (1)

Publication Number Publication Date
CN109675113A true CN109675113A (zh) 2019-04-26

Family

ID=66186185

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811470046.4A Pending CN109675113A (zh) 2018-11-26 2018-11-26 一种组织工程肌腱微组织的制备方法

Country Status (1)

Country Link
CN (1) CN109675113A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112741898A (zh) * 2019-10-29 2021-05-04 浙江大学 矿化肌腱胶原在制备修复骨-肌腱连接处的药物中的应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101332315A (zh) * 2007-06-29 2008-12-31 上海国睿生命科技有限公司 组织工程肌腱及其体外构建方法
CN101366976A (zh) * 2008-09-03 2009-02-18 陕西瑞盛生物科技有限公司 人源化异种细胞外基质材料及其制备方法
US20100124563A1 (en) * 2008-11-17 2010-05-20 Ingeneron, Inc. Biomatrix Composition and Methods of Biomatrix Seeding
WO2010101883A2 (en) * 2009-03-06 2010-09-10 Mayo Foundation For Medical Education And Research Tendon or ligament tissue engineering
CN102614546A (zh) * 2011-01-26 2012-08-01 香港中文大学 用于组织修复和生物人造组织工程的细胞片及其制备方法
CN105233342A (zh) * 2015-10-27 2016-01-13 泰山医学院 一种仿生多孔组织工程软骨支架制备方法
CN105999414A (zh) * 2016-06-20 2016-10-12 清华大学 制备人造微环境的方法及其应用
CN108245708A (zh) * 2016-12-28 2018-07-06 四川大学华西医院 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101332315A (zh) * 2007-06-29 2008-12-31 上海国睿生命科技有限公司 组织工程肌腱及其体外构建方法
CN101366976A (zh) * 2008-09-03 2009-02-18 陕西瑞盛生物科技有限公司 人源化异种细胞外基质材料及其制备方法
US20100124563A1 (en) * 2008-11-17 2010-05-20 Ingeneron, Inc. Biomatrix Composition and Methods of Biomatrix Seeding
WO2010101883A2 (en) * 2009-03-06 2010-09-10 Mayo Foundation For Medical Education And Research Tendon or ligament tissue engineering
CN102614546A (zh) * 2011-01-26 2012-08-01 香港中文大学 用于组织修复和生物人造组织工程的细胞片及其制备方法
CN105233342A (zh) * 2015-10-27 2016-01-13 泰山医学院 一种仿生多孔组织工程软骨支架制备方法
CN105999414A (zh) * 2016-06-20 2016-10-12 清华大学 制备人造微环境的方法及其应用
CN108245708A (zh) * 2016-12-28 2018-07-06 四川大学华西医院 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
严明明 等: "脂肪干细胞在肌腱组织工程中的研究进展", 《中南大学学报(医学版)》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112741898A (zh) * 2019-10-29 2021-05-04 浙江大学 矿化肌腱胶原在制备修复骨-肌腱连接处的药物中的应用

Similar Documents

Publication Publication Date Title
ES2520019T3 (es) Mejoras relativas a la descelularización de matrices de tejido para el implante de vejiga
CN105078613B (zh) 用于外科乳房手术的移植材料
CN106492285A (zh) 可注射的脱细胞软骨基质微粒及其在植入剂中的应用
CN102317445B (zh) 细胞、人工细胞构建体或三维复合组织装配体的冷冻保存方法
EP3040088A1 (en) Method for preparing an animal decellularized tissue matrix material and a decellularized tissue matrix material prepared thereby
Sano et al. Acellular adipose matrix as a natural scaffold for tissue engineering
CN102933705A (zh) 去细胞化的脂肪组织
Guasti et al. Chondrogenic differentiation of adipose tissue-derived stem cells within nanocaged POSS-PCU scaffolds: a new tool for nanomedicine
CN108310467A (zh) 一种组装型细胞衍生细胞外基质膜复合骨修复材料及其制备方法和应用
CN102266585B (zh) 一种用于女性盆底的生物复合补片及其制造方法
CN1306445A (zh) 植入用顺性脱水组织的制备方法
Ming et al. Repair of articular cartilage defects in rabbits through tissue-engineered cartilage constructed with chitosan hydrogel and chondrocytes
WO2018120672A1 (zh) 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途
Heuschkel et al. In vitro evaluation of bovine pericardium after a soft decellularization approach for use in tissue engineering
CN106492288A (zh) 可注射的脱细胞脂肪基质微粒及其在植入剂中的应用
CN102137926A (zh) 促进由成纤维细胞生成ecm和/或促进生物体系中成纤维细胞迁移
Lohan et al. Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix
CN109078226A (zh) 一种微孔隙脱细胞猪主动脉基质
CN102793593B (zh) 用于后巩膜加固术的异种巩膜片及其制备方法
CN105682697A (zh) 去除α-半乳糖的方法
CN104491927B (zh) 一种脱细胞舌基质材料及其制备方法
CN108653814A (zh) 一种脱细胞软骨基质材料的制备方法
CN109675113A (zh) 一种组织工程肌腱微组织的制备方法
CN106178107A (zh) 一种蚕丝/胶原复合支架及其制备与应用
CN108823154A (zh) 一种脂肪干细胞的无血清培养基及其制备方法

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20190426