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

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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
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tenocyte
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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
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Chinese PLA General Hospital
Taishan Medical University
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Taishan Medical University
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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。
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