Tissue engineering of articular cartilage: integration and remodeling of normal cartilages surrounding grafts and new cartilages

doi:10.3969/j.issn.2095-4344.2014.07.019

Abstract

Abstract:

BACKGROUND: Because the tissue regeneration capacity of articular cartilage is very low, the cartilage defects induced by joint injuries or degenerative diseases are hard to do their own repair.
OBJECTIVE: To comprehend the current status of tissue engineering of articular cartilage by analyzing data.
METHODS: The PubMed database and CNKI database from 2000 to 2013 were retrieved using computer with the key words of “articular cartilage, tissue engineering, signaling molecules, scaffolds” in English and “articular cartilage, tissue engineering, cell source, signaling molecules, scaffolds” in Chinese. A total of 202 literatures were screened out, and the repetitive and irrelevant researches were eliminated. Finally, 70 literatures were included for review.
RESULTS AND CONCLUSION: The most investigated cell sources for their potential in cartilage tissue engineering are chondrocytes and stem cells. Several cytokines, hormones and growth factors are known to influence the synthesis, anabolic and catabolic processes by chondrocytes. New biomaterial scaffolds in cartilage tissue engineering provide a comfortable niche for cells, where cells were stimulated to synthesize cartilage matrix, and to temporarily replace the function of the native matrix until new cartilage had formed. With the deepening of the researches, tissue engineering articular cartilage will exhibit good prospects.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: cartilage, articular, chondrocytes, tissue engineering, cytokines, cytoskeleton

CLC Number:

R318

Jiang Hui, Liu Xiao-zhou, Wang Rui . Tissue engineering of articular cartilage: integration and remodeling of normal cartilages surrounding grafts and new cartilages[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(7): 1094-1100.

Figures/Tables 1

2 细胞来源 Cell source 软骨组织工程技术理想的来源细胞是既易于分离、扩增，又可以合成大量的特定软骨基质成分，例如聚集蛋白聚糖、Ⅱ型胶原蛋白等关节软骨特定基质。软骨细胞和干细胞是当前软骨组织工程研究的主要细胞来源。 2.1 软骨细胞软骨细胞因在软骨组织中的存在部位不同，其形态亦异。近软骨表面是一些幼稚的细胞，体小呈扁椭圆形，细胞长袖与软骨表面平行，多为单个存在。越向深层，软骨细胞逐渐长大，变成圆形或椭圆形，在软骨的中央，软骨细胞成群分布，每群为2-8个细胞，它们都是由一个软骨细胞分裂而来，故称同源细胞群。在软骨组织工程研究中，软骨细胞是目前应用最为广泛的细胞来源。软骨细胞在体外能产生、重塑软骨细胞外基质，但自体软骨细胞的获取量毕竟有限，采集自病变关节的软骨细胞其细胞活性也相对较低。另外，单层软骨细胞扩增会降低细胞的分化能力，继而导致蛋白多糖合成及Ⅱ型胶原的表达降低，Ⅰ型胶原的表达增加[4-5]。软骨细胞寿命也是一项值得考虑的重要问题。在大多数软骨组织工程研究中，采用的软骨细胞多来自于尚未发育成熟的动物，这些实验动物的细胞增殖较快，而老年骨性关节炎患者来源软骨细胞的体外活性较低，相比骨性关节炎患者来源的软骨细胞其细胞分化潜能更佳[6-7]。尽管旋转生物反应器培养[8]，无血清培养[9]，低氧张力培养以及附加生长因子的培养等技术可以通过改变培养基条件弥补上述研究缺陷[10-11]，但研究表明，通过这些培养技术获得的软骨细胞并不利于软骨缺损的修复。体外培养自体软骨细胞的另一不利因素是细胞来源多来自于软骨病变周围，因此其软骨细胞的正常性能会明显低于正常软骨细胞。关节软骨只由一种特别的细胞类型组成，即软骨细胞，可以产生和维持软骨的细胞外基质，即胶原(大都为Ⅱ型胶原)和蛋白多糖(主要为聚集蛋白多糖)。近年来，国内不断有人采取自体软骨细胞移植、基质诱导的自体软骨细胞移植等方法治疗骨关节相关疾病，取得较佳疗效。组织工程化软骨移植修复程序主要包括四步即软骨移植前关节软骨活检、软骨细胞体外培养、细胞移植和软骨移植后功能锻炼。虽然软骨细胞的培养已相当成功，作为工程化软骨的细胞学基础，其来源尚未得到完全解决。从发展趋势上讲，从机体自身软骨组织获取及供体组织获取细胞数量均十分有限，不能满足工程化软骨形成的条件。大量的研究仍未从根本上解决工程化软骨所需软骨细胞数量和表型的稳定表达问题，限制了它的临床应用。 2.2 干细胞为了克服软骨组织工程中自体软骨细胞的缺陷，多能干细胞成为软骨细胞的另一重要细胞来源。用于体外分化软骨细胞的多能干细胞主要取材自骨髓和脂肪组织。骨髓源性干细胞易于获取，可分化为软骨细胞，即使在体外扩增后仍能保持其软骨细胞分化特性。多项研究表明，在各种3D培养环境中应用β型转化生长因子可提高骨髓间充质干细胞的软骨细胞分化性能[12-13]。在软骨组织工程中采用骨髓间充质干细胞作为细胞来源的主要缺陷在于骨髓间充质干细胞的增殖分化会导致细胞外基质堆积，继而造成骨髓间充质干细胞来源的软骨组织结构的机械性能显著低于软骨细胞种子来源的软骨组织[14]。Koga等[15]研究认为，这一问题可能与骨髓间充质干细胞在体外培养、扩增过程中增加了Ⅹ型胶原的表达有关，而Ⅹ型胶原为肥大软骨细胞的标志物。De Bari等[16]研究表明，Ⅹ型胶原表达于肥大软骨细胞，其肥大相关基因的表达会导致移植物血管形成增加，进而引起细胞死亡或钙化。此外，骨髓间充质干细胞会持续表达Ⅰ型胶原蛋白[17]，进而导致其软骨组织的基质成分有别于正常软骨组织。最近多项研究表明[18- 19]，通过抑制Ⅰ型和Ⅹ型胶原的表达有望控制骨髓间充质干细胞的分化途径，进而提高骨髓间充质干细胞来源软骨的组织性能。最近，多项研究表明，在3D培养基中加入维生素C，地塞米松和转化生长因子β等可使脂肪源性干细胞增殖、分化为软骨细胞，且可产生软骨特定基质成分，并会显著改善软骨组织的机械性能[20-21]。然而，脂肪间充质干细胞尽管能分化为软骨细胞，但其软骨形成潜能显著低于骨髓间充质干细胞，因此，尚需进一步的相关研究以提高脂肪间充质干细胞来源软骨细胞的软骨形成潜能。除了骨髓间充质干细胞和脂肪间充质干细胞，肌肉、滑膜以及骨膜等其他组织细胞也有望成为软骨组织工程的细胞来源，但这些组织来源的干细胞其软骨形成潜能远远低于骨髓间充质干细胞和脂肪间充质干细胞[22]，因此，在软骨组织工程中目前相关研究较少。 3 信号分子 Signaling molecule 多种细胞因子、激素类物质及生长因子会影响软骨细胞的合成及分解代谢过程。在以往软骨组织工程体外研究中[23]，转化生长因子β、胰岛素样生长因子、骨形态发生蛋白，成纤维细胞生长因子以及表皮生长因子等生长因子被用于提升软骨细胞表型，刺激细胞外基质产生，提高骨髓间充质干细胞的软骨细胞生成。骨形态发生蛋白家族成员中的骨形态发生蛋白2和骨形态发生蛋白7能促进骨髓间充质干细胞的软骨细胞形成，增加软骨细胞和骨髓间充质干细胞的基质产生。转化生长因子β超家族成员在软骨发生和修复中也起重要作用，目前多项研究表明，转化生长因子β1、转化生长因子β2、转化生长因子β3可促进软骨细胞增殖，增加细胞外基质合成[24-25]。另外研究显示，胰岛素样生长因子还能够刺激软骨细胞的合成代谢活性，诱导产生软骨细胞型细胞外基质[26]。 3.1 信号分子的联合效应目前多项研究表明，将多种生长因子加入软骨细胞或骨髓间充质干细胞体外培养基中会增加生长因子的生物学效应，例如胰岛素样生长因子/转化生长因子β1，胰岛素样生长因子1/转化生长因子β2，胰岛素样生长因子/骨形态发生蛋白2或胰岛素样生长因子/碱性成纤维细胞生长因子/转化生长因子β2等生长因子组合会增加软骨细胞的合成代谢，刺激细胞外基质生存增加[26-27]。但另有研究认为，胰岛素样生长因子/转化生长因子β，碱性成纤维细胞生长因子/转化生长因子β以及成纤维细胞生长因子2/胰岛素样生长因子等生长因子组合不会改善组织工程软骨的机械性能和组织学特性[28-29]。在另外一些研究中，还通过联合应用多种生长因子去诱导骨髓间充质干细胞的软骨细胞分化。Xiang等[30]采用胰岛素样生长因子和转化生长因子β1研究了其联合诱导骨髓间充质干细胞的软骨细胞分化过程。Kim等[31]和Im等[32]通过研究转化生长因子β2/骨形态发生蛋白7，转化生长因子β2/骨形态发生蛋白6，转化生长因子β2/骨形态发生蛋白2和转化生长因子β2/胰岛素样生长因子等生长因子组合促进骨髓间充质干细胞的软骨细胞形成过程发现，转化生长因子β2/骨形态发生蛋白7诱导骨髓间充质干细胞分化软骨细胞的效应最佳。另有一些研究显示，在单层和3D培养基中转化生长因子β3与骨形态发生蛋白2、骨形态发生蛋白4、骨形态发生蛋白6 以及胰岛素样生长因子联合应用可有效促进骨髓间充质干细胞的软骨细胞分化过程[33- 34]。 3.2 生物学效应的剂量和时间依赖性信号分子的生物学效应的产生除了依赖于其分子类型，还涉及应用时间、剂量等因素。Byers等[35]研究发现，相比持续应用转化生长因子β3，短时间应用转化生长因子β3可使软骨细胞水凝胶的葡萄糖胺聚糖(glycosaminoglycan，GAG)含量增加，并提升其压缩性能。在多数软骨组织工程研究中，转化生长因子β，成纤维细胞生长因子2和骨形态发生蛋白等生长因子的常用浓度为 10 µg/L[23]，但Byers等研究发现，在软骨细胞培养基中连续加入1，2.5，5，10 µg/L 转化生长因子β会环比提升组织的物理和生物学性能。 3.3 编码生长因子基因转染软骨细胞外源性生长因子半衰期短，局部使用很快被稀释和代谢，需反复使用，价格昂贵，且治疗剂量相对较大，从而带来许多相应的问题。随着转基因技术的发展，可在不影响细胞特殊表型的前提下，将生物活性物质基因转入种子细胞中，通过基因表达产生内源性生物活性物质，调控自身增殖分化。在这方面国内学者也做了大量工作。赵建宁等[36]利用低相对分子质量壳聚糖制备了一种新型载基因纳米微粒作为基因转移系统，进行转染兔关节软骨细胞的研究，评估了其体外转染软骨细胞的性能，研究结果表明，壳聚糖可以和目的基因质粒相互作用形成带有表面正电荷的纳米微粒，且能在很大程度上保护质粒DNA不受DnaseI的降解，对细胞毒性小，转染软骨细胞后基因能够有效表达，他们认为载基因低相对分子质量壳聚糖纳米微粒能够有效地转染软骨细胞，是一种很有潜力的纳米局部传输系统。王瑞等[37]通过新西兰大白兔膝关节全层软骨缺损模型，采用一种靶向软骨的非病毒纳米基因传递系统，携带增强型绿色荧光蛋白在体内外对软骨细胞进行转染，探讨其促进软骨修复的方法低相对分子质量壳聚糖/DNA纳米复合物可以有效地转染体外单层培养的软骨细胞和软骨组织片，在体内应用时，低相对分子质量壳聚糖/增强型绿色荧光蛋白基因纳米复合物可以安全有效地转染软骨细胞，并表现出一定的靶向性。 3.4 机械刺激的生物学效应为了改善组织工程软骨的机械性能，机械刺激因素也是当前研究的一个方向，由此各种生物刺激器得以研制成功[38]，以对细胞-种子结构施以机械载荷，继而提高软骨组织结构的力学性能。在软骨组织工程学研究中，直接动态或自由加压以及流体静力加压是目前研究最多的两组力学加载方式。多项研究表明，直接动态加压应用于软骨细胞-种子结构会诱导细胞外基质产生和/或软骨细胞增殖[39-40]。最近研究显示，相比非加载对照组，动态加压作用于骨髓间充质干细胞-种子结构可刺激软骨样细胞外基质成分增生[41-42]。两项体外实验研究表明，流体静力加压可改善组织工程软骨的机械性能，但数据结果很大程度上依赖于应用的载荷参数[43- 44]。除了对细胞代谢活性的影响，流体静力加压还能够刺激影响体外软骨细胞的表型[45]。此外，最新研究显示，流体静力加压还可用于刺激骨髓间充质干细胞[46]、脂肪间充质干细胞以及滑膜源性干细胞向软骨细胞分化、增殖[47-48]。除了上述载荷方式，剪切载荷、滑动/滚动切痕载荷、拉伸载荷、离心及重力加载方式也被用于软骨组织工程的研究中[49-50]。在将来的软骨组织工程学研究中，除了要进一步考察特定机械因素的作用外，不同机械刺激的联合效应以及相关的刺激参数也是未来的一个研究方向。 4 支架 Scaffolds 在软骨组织工程研究中，生物材料支架的应用目的是为细胞提供一个适宜的环境，刺激细胞软骨基质合成，临时替代天然软骨基质的功能，直至新生软骨形成。为了实现这一生物学功能，用于软骨组织工程研究的生物支架应具备如下特征：①可控性生物降解能力，且不产生毒副产物。②多孔性，以利于营养物质和废物的扩散。③维持细胞活性、分化、增殖以及细胞外基质产生的能力。④缺损部位的组织固定和整合能力。⑤对组织工程软骨的机械支持性能等。根据以上特点，多种天然和生物合成聚合物被用作软骨组织工程的支架材料。 4.1 支架类型用于支架材料的天然聚合物包括多种蛋白类(例如蛋白丝、纤维以及胶原等)，糖类，例如琼脂糖、透明质烷以及聚氨基葡糖等。这些物质均以含水量较高的水凝胶形式用于组织工程学研究，可被制成可注射的流体形式，进而与软骨细胞很好的混合。水凝胶结构的最大优点在于软骨细胞可支架包裹后能够维持其自然的软骨细胞表型，这对于研究其组织工程软骨细胞的机械载荷刺激十分有利。另外，多项研究表明，天然支架材料能随着组织结构的成熟促进天然细胞外基质的重塑[51-52]。目前，在软骨组织工程学研究中，应用最为广泛的人工合成聚合物支架是聚-α-羟基酯，尤其是聚乳酸和聚乙醇酸，其具备的生物可降解能力，在美国已经获食品和药品监督管理局(US Food and Drug Administration，FDA)批准进入临床应用阶段。这些合成支架材料相比水凝胶其机械强度更佳，更易于固定到缺损部位，改善软骨修复处的载荷性能[53]；另外，通过技术手段，还可以对这些支架材料的降解性能，结构和机械强度等生物学特性进行修饰。合成支架材料的不足在于细胞常常不能维持其软骨细胞表型，且产生的细胞外基质性能较差[54]。 4.2 支架材料的结构、孔隙度及刚度支架材料的多孔性、孔径大小以及互联性十分重要，可对细胞迁移、氧弥散，营养维持、废物排出以及信号分子的作用产生显著影响[55]。多项研究显示，从细胞-种子结构外围向中心的氧运送会导致组织结构中心区域的细胞死亡[56-57]。另外，支架材料的多孔性还可以改善植入物与周围天然软骨的机械连锁作用，使连接界面之间获得更大的稳定性。最近研究表明，生物支架的孔隙度和参透性会显著影响软骨细胞的表型和增殖能力[58]。Lien等[59]研究发现，100-500 μm的支架孔径对于促进细胞增殖能力是最佳的。利用支架结构和孔隙度特征还可诱导生成特定组织结构形态。Woodfield等[60]在其软骨组织工程学研究中发现，应用带有空隙梯度的100%互联多空结构支架可促进葡萄糖胺聚糖和Ⅱ型胶原不均分布和带状分布。支架材料的刚度会对种子细胞的机械环境产生影响，进而影响培养基中的细胞增殖和组织生长。Genes等[61]研究表明，基质刚度的增加会显著影响软骨细胞的形态，基质刚度增加后会使脆弱的圆形肌动蛋白的转变为硬实的扁平形态。但基质刚度的增加对于组织工程软骨未必有利，例如Ng等[62]研究显示，由于基质产物更有效的保留，高浓度琼脂糖(3%)能够导致组织结构的初始刚度更大，但其组织的长期性能会显著低于2%琼脂糖组织。 4.3 支架的生物降解能力目前，在软骨组织工程学研究中应用的支架材料基本均具备生物可降解能力。支架材料的在时间和空间上的可控降解性能的会影响新生软骨组织的产生和基质沉积。最佳的降解动力性可保证支架的初始稳定性和形态特征，同时不应妨碍新生软骨细胞外基质的沉积。多项研究表明，相比快速降解的支架材料，降解速度较慢的支架会导致更多的细胞外基质沉积[63-64]。另外，Bryant等[65]研究发现，支架的降解能够有利于新生软骨组织与移植物周围天然软骨的重塑和整合。为了提高支架的降解速度，水解作用类物质[65]，基质金属蛋白酶(matrix metalloproteinase，MMP)敏感类肽以及多种外源性酶类被用于软骨组织工程学研究中[66-67]。Ng等[67]研究发现，采用琼脂水解酶控制琼脂糖支架降解速度后，随着培养时间的延长，会引起软骨组织的胶原含量增加，机械性能增强，他们推测这是由于支架降解速度加快后，组织的营养支持增加和胶原纤维生成的空间增大所致。另外，Chung 等[68]研究发现，负载骨髓间充质干细胞的筛孔水凝胶会随着支架的降解速度增加，而使组织的葡萄糖胺聚糖和Ⅱ型胶原含量升高，继而使其软骨组织的机械性能显著优于非水凝胶支架组织。"

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