Application of mesenchymal stem cells in articular cartilage tissue engineering

doi:10.3969/j.issn.2095-4344.2014.45.023

Abstract

Abstract:

BACKGROUND: The development of tissue engineering techniques provides new methods and ideas for the repair and functional reconstruction of articular cartilage defects.

OBJECTIVE: To summarize the research progress in the application of mesenchymal stem cells, as seed cells, in articular cartilage tissue engineering.

METHODS: A computer-based retrieval was performed to search articles describing articular cartilage tissue engineering and mesenchymal stem cells published between January 1st, 2000 and September 30th, 2014 in PubMed database with the key words of “articular cartilage defects; cartilage tissue engineering; mesenchymal stem cells” in English. Seventy articles were retrieved initially, and only 49 were included in further analysis.

RESULTS AND CONCLUSION: The ability of the articular cartilage for defect self-repair is limited, and current clinical treatments cannot be satisfactory. Development of tissue engineering provides a new idea for problem-solving. In the selection of seed cells, chondrocyte is limited to dedifferentiate, and embryonic stem cell is restricted by ethical, legal and other aspects. Autologous mesenchymal stem cells, which are easy to be amplified and exhibit excellent cartilage differentiation potential, have gained widespread attention. But there is still some controversy on the current application of tissue engineering techniques for repair of articular cartilage defects, including a certain gap between the long-term effects and the clinical applications. So the effect of mesenchymal stem cells on biological structure and mechanical function still needs further studies.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: mesenchymal stem cells, cartilage, articular, tissue engineering

CLC Number:

R318

Zhang Yong-xing, Zhao Qing-hua. Application of mesenchymal stem cells in articular cartilage tissue engineering[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(45): 7342-7347.

Figures/Tables 1

2.1 骨髓间充质干细胞成人骨髓间充质干细胞是最广泛应用于软骨组织工程构建研究中的间充质干细胞，被认为是骨肌系统构建的“金标准”。骨髓间充质干细胞容易通过骨髓穿刺获得，具有在体内和体外分化为结缔组织如软骨、骨和脂肪组织的能力。使用骨髓间充质干细胞的主要优点在于为组织构建提供了自体细胞来源，避免了潜在的免疫排斥。自体和同种异体的骨髓间充质干细胞有免疫抑制能力[9]。Wakitani等采用体外培养的兔自体骨髓间充质干细胞修复关节软骨全层缺损后，组织学评分发现术后3个月内缺损被软骨组织所修复，其后他们又进行了人关节软骨缺损的修复，也获得类似效果。但经长期观察发现，已形成的透明软骨最终被纤维软骨和骨样组织所代替。并且，由于骨髓间充质干细胞具有多向分化的潜能，故干细胞中只有一小部分能自发地分化为软骨祖细胞，其在体外的分化很大程度上依赖于特殊的培养条件，诱导因子在骨髓间充质干细胞的定向分化中起到非常关键的作用。Kuroda等[10]研究显示，自体骨髓间充质干细胞不存在免疫排斥反应，可成为理想的软骨种子细胞来源。Rowland等[11]发现用细胞外基质作为支架材料，如去矿化的骨基质，不仅可以提供良好的生物力学支撑，还可以调节细胞行为、减少免疫反应，改善体内软组织修复的长期效果。除此之外，细胞因子对于骨髓间充质干细胞成软骨分化能力也有一定的影响[12]。Wei等[13]发现骨髓间充质干细胞合成的细胞外基质和转化生长因子β3均能存进软骨损伤处的骨髓凝块向软骨方向分化。Bhumiratana等[14]通过转化生长因子β诱导骨髓间充质干细胞模拟体外间充质凝结的过程，构建了一种生物机械性能好(弹性模量> 800 kPa，均衡摩擦系数< 0.3)的生物软骨，用于软骨缺损修复，取得了比较好的效果。不少学者试图通过改良支架材料性能复合骨髓间充质干细胞来达到更好的修复效果。Bai等[15]和Tan等[16]分别发现，生物活性玻璃和三维多孔支架表面改性复合骨髓间充质干细胞比起自体骨组织，能够更好地在宿主体内进行整合，在移植物表面形成透明软骨样组织，表达更多的Ⅱ型胶原。Zheng等[17]和Xie等[18]分别发现，仿生结构的聚乳酸-羟基乙酸(PLGA)和聚己酸内酯(PCL)多孔海绵支架对骨髓间充质干细胞均具有良好的亲和性；Ahmed等[19]发现新鲜的血纤维蛋白和富血小板的纤维蛋白胶与骨髓间充质干细胞复合均能收到良好的效果，血纤维蛋白的效果似乎要好过富血小板的纤维蛋白胶；Duan等[20]用温度梯度介导、热诱导相分离的方法来编织细胞外基质，后与骨髓间充质干细胞复合植入裸鼠皮下，与仅仅通过冷冻干处理的支架相比，前者的压缩模量更高，机械性能更佳；同时，支架组成材料的有序排列对于软骨缺损的修复也十分重要[21]。众所周知，软骨损伤部位往往会产生大量的炎性因子，如白细胞介素1，这些炎性因子会影响细胞材料复合体的机械性能。Ousema等[22]发现3D网状支架可以保持材料良好的机械性能，从而减少了炎性因子对材料细胞复合体的负面影响。软骨组织工程移植物通常能够保持接近正常的抗压能力，但是其抗拉伸能力、不均一性和各向异性都不如正常软骨组织。为此，Huang等[23]设计了一种新型的滑动接触生物负荷反应器，通过增加两关节接触面的生理负荷来刺激其机械性能的改善。Erickson等[24]通过不同浓度(1%，2%，5%)的丙烯酸酯透明质酸交联水凝胶高聚物复合骨髓间充质干细胞构建组织工程复合物，发现随着高聚物密度的增加，骨髓间充质干细胞成软骨分化能力和基质形成能力得到增强，但机械性能却随之下降，在1%浓度的复合物中，其均衡压缩模量为0.12 MPa，糖胺多糖含量接近湿质量的3%，分别达到了正常组织的25%和50%。也有人通过技术方法与流程的革新来提高软骨组织工程的有效性。Shi等[25]通过聚L-乳酸-乙醇酸(PLLGA)支架与微骨折法结合，将软骨缺损腔与软骨下骨髓钻通，使得骨髓间充质干细胞可以直接进入缺损部位，从而将体内取材、体外培养、体内回植的过程转变成“一步”。Wise等[26]通过静电纺丝复合聚己酸内酯纳米支架(500-3 000 nm孔径)构建软骨组织工程支架替代细胞外基质获得了成功。近年来，研究者们发现软骨组织工程不应紧紧局限于表层软骨，还应关注软骨下骨的修复，尤其是对于高负荷区的骨软骨缺损[27]，于是提出了骨软骨修复的概念[8]。Da等[28]通过研究发现致密层在骨软骨修复中具有重要作用，含有致密层的双相支架的机械性能(包括抗拉伸和抗剪切能力)更强；体内试验也表明，其表观评分、糖胺多糖和胶原含量、显微断层成像效果以及组织学性质更佳；Liu等[29]和Zhou等[30]分别通过Ⅰ型胶原和聚羟基乙酸羟基磷灰石双向支架复合骨髓间充质干细胞用于软骨组织工程均取得了满意的效果。 2.2 脐带间充质干细胞对骨髓间充质干细胞创造性的研究为关节软骨缺损的修复提供了新的种子细胞来源，成为目前组织工程修复关节软骨缺损研究的主要细胞类型，但由于对骨髓间充质干细胞分化的机制并不明确，且这种分化并不稳定、分化率有限、分化后的软骨细胞显示出易退化和衰老的特性，在软骨缺损修复长期观察中发现暂时形成的透明软骨最终会被纤维软骨或纤维组织所替代，这些局限性都限制了骨髓间充质干细胞在软骨组织工程中的进一步应用；在研究中科学家们发现，人类脐带来源的间充质干细胞似乎可以弥补骨髓间充质干细胞的不足。脐带间充质干细胞是一种多能基质细胞，它具有可黏附性和可塑性，细胞表面一样可以产生像其他间充质细胞表面产生的标记分子，如CD73、CD90和CD105，而且不是造血细胞。作为一种胚外细胞来源，脐带间充质干细胞比骨髓间充质干细胞有更快的增殖速度，数量翻倍所用时间更短[31]。比起骨髓间充质干细胞，脐带间充质干细胞在体外保持增殖分化特性的时间要更长[32]。细胞采集之后，脐带间充质干细胞经过7次传代，已经达到了具有细胞数量300倍增长，而始终保持分化潜能的特性[33]。然而，脐带间充质干细胞的成软骨细胞分化能力的研究得出的结论却不一致。在Wang等做的实验中，未经转化生长因子β1诱导的脐带间充质干细胞在黏多糖检测和Ⅱ型胶原的检测中呈阳性。而在Karahuseyinoglu等[34]做的相同实验中，脐带间充质干细胞和骨髓间充质干细胞均只检测到少量的Ⅱ型胶原。在对脐带间充质干细胞和骨髓间充质干细胞所做的实验中都检测到了Ⅰ型胶原，而拥有更好丝状细胞外基质的脐带间充质干细胞组中观察到比骨髓间充质干细胞组更大的胶原颗粒。 2009年，研究者将骨髓间充质干细胞和脐带间充质干细胞在几乎完全相同的条件下传代培养，然后接种到聚乙醇酸支架上，在成软骨基质中培养[35]。这次实验的主要成果在于人们发现，脐带间充质干细胞产生的胶原数量要高于骨髓间充质干细胞(以每平方单位基质和每个细胞为单位来计数)。2010年，人们将骨髓间充质干细胞和脐带间充质干细胞在几乎完全相同的条件下又进行了一次对比实验，这次实验是将它们接种在以微球体为基质的倾斜支架上[36]。在这次骨软骨构建实验中，脐带间充质干细胞在胶原合成和碱性磷酸酶活性方面要高于骨髓间充质干细胞，然而在Sox9和Runx2的基因表达方面，前者仍逊于后者。总体来讲，脐带间充质干细胞在分化能力方面要逊于骨髓间充质干细胞，但是比后者有更强的组织形成能力。但必须指出的是，这些发现仅适用于特定的体外环境，如果脐带间充质干细胞确实能够对体内诱导信号做出比较积极响应的话，脐带间充质干细胞可能在组织再生的质量和成功率方面有更大的骨软骨再生的潜力。 2.3 脂肪间充质干细胞骨髓间充质干细胞虽然可以作为关节软骨组织工程的主要种子细胞，但取材困难、原代数量少仍然是限制其应用的“瓶颈”。作为替代，脂肪来源的间充质干细胞逐渐引起了大家的兴趣。脂肪组织容易获得，而且间充质干细胞的含量高(约占间充质细胞总数的5%)，在同等条件下是骨髓间充质干细胞含量100倍[37]。脂肪组织可以通过脂肪吸引术这种微创技术大量获得，继而获得大量的脂肪间充质干细胞[38]。脂肪间充质干细胞成软骨分化涉及3个时相，即凝结、增殖、分化；Zhang等[39]发现脂肪间充质干细胞在未经诱导的情况下也能修复全层透明软骨缺损；Merceron等[40]用一种可注射的自固化纤维素水凝胶复合脂肪间充质干细胞进行成软骨分化培养，效果与骨髓间充质干细胞并无明显差异。Buckley等[41]发现在含有转化生长因子β3的环境下，低氧能够促进髌骨下脂肪垫来源的间充质干细胞形成细胞外基质；然而，在正常氧浓度和转化生长因子β3存在的情况下，软骨细胞复合的组织工程支架却能产生最佳机械性能。Jakobsen等[42]比较了骨髓间充质干细胞和脂肪间充质干细胞复合透明质酸支架在软骨组织工程中的应用，结果发现，3周后，骨髓间充质干细胞表达Ⅱ型胶原mRNA的水平是脂肪间充质干细胞的600倍；而Ⅰ型胶原mRNA和蛋白聚糖的表达二者基本相同，这一结果表明，在软骨组织工程应用方面，骨髓间充质干细胞似乎比脂肪间充质干细胞更具优势。 2.4 其他来源的间充质干细胞与基因转染除了以上来源，骨膜、关节滑膜也是常见的间充质干细胞来源。2001年，De Bari等从人关节周围的滑膜中分离出了间充质干细胞，即滑膜间充质干细胞。滑膜中的间充质干细胞比例很高，平均每毫克滑膜组织中含(3.0-4.0)×103个有核细胞，平均10个有核细胞中就含有8个左右间充质干细胞，因此只需少量的标本就可以获得足量滑膜间充质干细胞，其数量是骨髓来源间充质干细胞的100倍以上，并且随着供体年龄的增加，间充质干细胞数量也并未出现明显减少。Iwakura等[43]发现转化生长因子β1和骨形态发生蛋白7的联合或序贯使用能够促进滑膜来源间充质干细胞的成软骨分化和浅表层蛋白的分泌。Hermida-Gómez等[44]研究发现，骨关节炎患者的滑膜间充质干细胞表达CD271要高于健康人的滑膜间充质干细胞，且更多地参与关节软骨的修复，修复效果更好，质量更高。在增殖能力方面，脂肪来源的间充质干细胞在第7代即丧失增殖力，而滑膜来源间充质干细胞在10代后仍保持增殖能力[43]。这些发现都在一定程度上拓宽了间充质干细胞的取材来源。为解决种子细胞来源困难的难题，学者们将转基因技术应用到关节软骨组织工程，将转化生长因子、骨形态发生蛋白、碱性成纤维细胞生长因子、胰岛素样生长因子等基因借助载体转染至相应靶细胞，使之分化为软骨细胞，其中应用最多的靶细胞是骨髓间充质干细胞。Cao等[45]用腺病毒介导将Sox-9基因转移到兔骨髓间充质干细胞中，发现骨髓间充质干细胞成软骨分化能力增强。Bai等[46]发现骨形态发生蛋白7基因转染的骨髓间充质干细胞分泌Ⅱ型胶原、糖胺多糖量增加，与阳性对照组(转化生长因子β1、地塞米松诱导)几乎相同。Hu等[47]用抗凋亡蛋白基因Bcl-xL，Elsler等[48]用北美萤火虫荧光素酶、β-大肠杆菌半乳糖苷酶和人胰岛素样生长因子1对间充质干细胞进行基因转染，发现转染细胞生存率高，细胞毒性小，且在长期培养中软骨分化能力不受影响。但Seo等[49]发现转染基因序列对宿主体内本身的基因结构造成了破坏，且长期的生长因子过表达会导致干细胞发生一系列的改变。除此之外，间充质干细胞与转染生长因子后的软骨细胞共培养也是不错的替代方式。关于基因转染的可行性与安全性，还需进一步的探讨。"

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