In situ tissue engineering in the field of bone and cartilage repair: application and problems

doi:10.3969/j.issn.2095-4344.1149

Abstract

Abstract:

BACKGROUND: In situ tissue engineering technology has obvious advantages compared with the traditional tissue engineering technology and exhibits good effects in promoting tissue regeneration and bone/cartilage repair.

OBJECTIVE: To review the application and problems of in situ tissue engineering technology in bone/cartilage repair

METHODS: A computer-based search of CNKI, PubMed, and Elsevier was performed for relevant articles concerning the application of in situ tissue engineering in the field of bone and cartilage repair published from January 2000 to November 2018. The keywords were “in situ tissue engineering, bone, cartilage, bone defects, cartilage repair, tissue engineering” in Chinese and English, respectively. The article types were review, basic and clinical studies.

RESULTS AND CONCLUSIONS: In situ tissue engineering technology is a technology developed for regeneration and restoration based on traditional tissue engineering technology, which has achieved good results in the repair of bone injury. It does not rely on exogenous seed cells and has the ability to repair bone damage by various new scaffold materials which possesses unique structure and physicochemical properties. These scaffold materials can recruit stem cells around the damaged site to migrate to the injured site and complete the in situ repair of bone damage. In situ tissue engineering technology provides new ideas and directions for cartilage damage repair, and has new application value in cartilage injury repair. The main purpose is to use the implanted scaffold material to induce the local microenvironment, and induces the peripheral cells to migrate to the lesion site to play a repairing role.

Key words: in situ tissue engineering technology, bone, cartilage, repair, tissue engineering, scaffold materials, cytokine, in situ repair of injury

CLC Number:

R459.9|R318.19

Zhang Jun, You Qi, Zou Gang, Ge Zhen, Liu Yi. In situ tissue engineering in the field of bone and cartilage repair: application and problems [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(20): 3255-3260.

Figures/Tables 1

2.1 原位组织工程技术的概述原位组织工程技术又被称为原位诱导再生技术，是指不使用传统组织工程技术所需要的外来种子细胞，通过性能良好的支架材料与体内微环境的相互作用，促进并诱导自体干细胞增殖、迁移并黏附在支架材料上，进而实现损伤组织的原位再生[21]。原位组织工程技术具有以下特点[21]：利用自体细胞特别是干细胞进行组织修复，避免了体外分离培养种子细胞的繁琐过程，并且避免了体外培养种子细胞容易污染的缺点；由于该过程没有添加外源性种子细胞，因此极大程度地避免了免疫排斥反应；通过一定的手段使原位细胞迁徙到病变部位，从而达到修复效果，该过程更加接近于人体损伤组织的自我修复，将生物体视为完整的生物反应器，这样的修复效果或许是更佳的。由于原位组织工程技术不涉及对体外细胞和材料进行广泛操作来产生功能化的组织，而是借助外在支架材料来诱导受损部位的自行修复，因此最终修复的组织与机体本身具有很好的相容性和适应性，并且远期功能也有一定的保障。原位组织再生的成功依赖于：将宿主干细胞或祖细胞有效募集到植入生物材料支架中，并将浸润细胞诱导成组织特异性细胞谱系，以用于功能性组织再生。 2.2 原位组织工程技术的3大要素 2.2.1 种子细胞(宿主细胞) 虽然原位组织工程不需要外源的种子细胞，但仍需要招募体内自体的内源性细胞作为种子细胞，参与损伤修复。其中，骨髓间充质干细胞是最先应用于组织工程研究的种子细胞[22]，由于其具有较好的增殖能力及较强的多向分化能力，因此在组织工程领域得到了广大学者的青睐。相关研究表明，当创伤应激发生时，机体可动员体内的骨髓间充质干细胞进入外周血，然后经过复杂的过程最终归巢到损伤部位，进行损伤组织修复[23]。在骨和软骨损伤过程中，骨髓间充质干细胞是最容易被募集的一种干细胞，并且具有较强的成骨和成软骨作用，因此成为骨和软骨损伤修复过程中首选的种子细胞[24-25]。如何促进支架材料的血管化，是原位组织工程技术的关键，也是难点，虽然目前很多学者通过募集内皮细胞到受损组织，使其向血管分化，但是内皮细胞存活力低、黏附性差且易老化[26-28]。因此，学者们又将目光转移至内皮祖细胞。内皮祖细胞是内皮细胞的前体细胞，主要分布在骨髓中，由于其具有较好的分化能力和增殖能力，因此很多研究者将其作为原位组织工程技术的种子细胞[29-30]。此外，也有研究者将骨膜来源的细胞应用于骨组织工程中，通过诱导它来促进损伤部位的修复[31-32]。 2.2.2 支架材料支架材料是原位组织工程的关键部分，因为其结构可用于模拟体内环境，作为细胞及细胞因子的载体引导细胞生长和浸润，并将细胞募集到损伤部位，让细胞在受损部位发挥作用；此外，支架材料还能将细胞和细胞因子局限在受损部位，防止其随着液体的外流而失去相应效果。其中，支架材料的结构尤其重要，主要是孔隙率和微观结构，生物材料的孔隙率和微观结构可调节细胞浸润和炎症反应，因此目前常采用多种技术来改变生物材料的结构和孔隙率[33-34]。当今应用于原位组织工程中的支架材料主要分2大类：合成高分子材料和天然高分子材料，合成高分子材料主要有聚羟丁酯、聚乳酸和聚乙醇酸等[35]，天然高分子材料主要有壳聚糖、丝素蛋、纤维素等[36]。 2.2.3 细胞因子细胞因子是原位组织工程技术的又一大核心要素，其能够促进细胞的增殖分化，诱导细胞趋化、迁徙，此外还能够与细胞膜上特异性的分子受体结合，增强细胞的黏附作用。目前研究发现，多数细胞因子与干细胞趋化有着密切关系，其中基质细胞衍生因子是目前研究得最多的一种趋化因子[37]。Liu等[38]实验证明，基质细胞衍生因子1在促进骨损伤的内源性修复方面发挥重要作用。此外，还有诸多细胞因子具有趋化作用，在原位组织工程技术中发挥重要作用[39-42]，如：胸腺表达趋化因子(又称胸腺趋化因子25)、单核细胞趋化蛋白1、血管内皮生长因子、血小板衍生生长因子等。但是，由于原位组织工程领域中所使用的细胞因子在体内半衰期较短，很容易降解，因此限制了这些细胞因子对体内细胞的招募和迁移作用。所以，很多研究者通过使用一些物理和化学方法改变细胞因子在生物体内的状态，来促进其在体内的长期有效释放[43-47]。 2.3 原位组织工程技术在骨损伤修复领域的应用骨缺损一直是临床的一大难题，困扰着当今的临床医生。随着组织工程技术的快速发展，用于骨损伤修复的相关技术得到了新的提高。最近几年，原位组织工程技术应用于骨损伤修复的研究不断深入，并取得了良好的效果。原位组织工程技术修复骨缺损最关键的问题就是寻找一种骨特异性支架，这也是当今的一大难点。骨特异性支架材料既要具有较强的机械性能又要具有最小的免疫和炎症反应，同时还能招募损伤部位周边细胞的迁移并能够促进细胞的增殖和分化。目前应用于原位骨再生的生物材料包括磷酸钙、硫酸钙和羟基磷灰石。目前很多研究证实，基质细胞衍生因子1α在骨修复方面具有很重要的作用，其能够募集内源性祖细胞和干细胞迁移到损伤部位，从而能进行组织损伤的修复和再生[48]，将基质细胞衍生因子1α与不同支架材料复合促进骨缺损的原位修复已取得了较大成就。 Shi等[49]在体外将基质细胞衍生因子1α与胎牛脱钙骨相结合，构建了功能化骨替代物，该复合支架材料能够不断、可控的释放基质细胞衍生因子1α，通过动员干细胞来修复缺损骨组织。将该复合支架材料植入大鼠股骨缺损模型中，发现植入后3 d能够有效动员CD34+和c-kit+内源性干细胞归巢到受损部位，micro CT结果显示受损部位的骨矿化和骨积累明显增加，并且还发现骨保护素和骨桥蛋白早期表达明显上调，成骨细胞数量也很快增加。Eman等[50]将低浓度的基质细胞衍生因子1a、明胶和生物陶瓷复合制备了复合生物材料支架，通过植入裸鼠皮下并在植入后6周后取材发现：成骨相关指标明显升高，如Ⅰ型胶原、骨钙素等，并且还意外的发现该复合支架具有成血管的作用。该研究首次证明了低浓度基质细胞衍生因子1a可有效募集内源性细胞，并且募集的细胞具有成骨分化潜能。Niu等[51]成功制备了二氧化硅渗透的三维胶原支架，该支架具有高度的生物相容性和可降解性，然后将基质细胞衍生因子1a与该支架复合并移植到小鼠体内，6周后发现该复合支架具有异位成骨和毛细血管生成的作用。骨形态发生蛋白能够诱导间充质干细胞成骨分化，同样也具有招募受损部位周围干细胞迁徙并向骨分化的能力[52]。此外，还有很多细胞因子可与不同的支架材料复合，诱导周边干细胞向受损的部位迁移并进一步成骨分化，达到修复骨缺损的目的[53]。 2.4 原位组织工程技术在软骨损伤修复领域的应用由于软骨没有血管和神经，因此损伤后很难自行修复，即使使用手术或者其他办法治疗后仍不能得到很好的修复。原位组织工程技术的出现，为软骨损伤修复提供了新的思路，同时也弥补了传统组织工程技术的某些缺陷。原位组织工程技术软骨修复的机制主要是：通过植入可降解的多孔支架材料，使其在体内能够诱导自体干细胞迁移并聚集在缺损软骨区域，并且最终分化为软骨细胞，实现软骨修复。 Filová等[54]构建了一种新型的无细胞支架材料，该支架将聚乙烯醇纳米纤维和透明质酸/Ⅰ型胶原/纤维蛋白水凝胶复合，其中聚乙烯醇纳米纤维里面包含有脂质体、碱性成纤维细胞生长因子和胰岛素，通过控制性释放使碱性成纤维细胞生长因子和胰岛素这些细胞因子持续发挥作用，释放出的细胞因子能够从骨髓中募集间充质干细胞。 Chen等[55]制备了一种胶原支架材料，该支架材料具有良好的机械性能，并且能够在体外诱导骨髓间充质干细胞在支架中的迁徙。随后将基质细胞衍生因子1与该支架复合来进一步促进其诱导细胞迁徙的能力，并将该复合支架植入兔软骨缺损模型中，术后6，12周取出标本观察软骨修复情况，实验发现该复合支架材料具有促进软骨再生和修复的作用，最终形成了类似软骨的结构。其机制主要是该复合支架材料能够募集软骨损伤部位周围的骨髓间充质干细胞，发挥软骨损伤修复作用。 Cook等[56]也通过将骨形态发生蛋白7与支架材料复合，修复犬股骨髁软骨损伤，结果显示该软骨损伤模型得到了很好地修复，其主要也是通过诱导内源性骨髓间充质干细胞来发挥修复作用的。此外，微骨折手术是当今治疗软骨损伤常用的一种手段，主要是使软骨下骨发生微骨折，让骨髓腔中的间充质干细胞进入关节腔来进行原位损伤修复。但是，单纯的微骨折手术用于软骨损伤修复目前也面临着很多的问题和不足，因此近几年来很多学者通过将负载有细胞因子的支架材料，与微骨折技术联合来促进软骨损伤的原位修复，并取得了很不错的结果[57-58]。"

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