Relevant cell growth factors in tissue engineering ligament/tendon construction

doi:10.3969/j.issn.2095-4344.2013.07.025

Abstract

Abstract:

BACKGROUND: Construction of tissue engineering ligament/tendon is becoming a hot spot in ligament-reconstructive research recently, which is closely associated with cytokines in vitro or in vivo.
OBJECTIVE: To search suitable cytokines for tissue engineering ligament/tendon construction through analyzing and summarizing the current research status of growth factors since 2001, and to explore the action mechanism of these cytokines.
METHODS: A computer retrieval was performed in VIP Periodical Full-Text Database and PubMed database for articles about tissue engineering ligament and relevant cytokines from January 2001 to December 2011 using the key words of “ligament, tendon, tissue engineering, growth factor” in Chinese and English, respectively. Literatures that are repetitive in the content and irrelevant with the objective were excluded. Finally, 33 literatures were involved in further analysis.
RESULTS AND CONCLUSION: There are many studies about the growth factors related to tissue engineering ligament/tendon, and most of them can improve the proliferation and differentiation of seedcells into tendon cells, strengthen the biomechanical activity of tissue engineering tendon, and reduce scar formation. Local injection or transgenosis can help the cells to express target proteins efficiently. Application of cell growth factors in tissue engineering ligament/tendon construction is of great significance.

Key words: tissue construction, academic discussion of tissue construction, ligament, tendon, tissue engineering, growth factors, scar, the National 863 Program of China

CLC Number:

R318

Cai Tao-yi, Chen Xiong-sheng, Zhou Sheng-yuan, Wang Hui, Sun Yan-qing, Dong Xing-cheng. Relevant cell growth factors in tissue engineering ligament/tendon construction[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(7): 1289-1294.

Figures/Tables 1

2.1 碱性成纤维细胞生长因子碱性成纤维细胞生长因子是重要的有丝分裂促进分子，肌腱愈合过程中碱性成纤维细胞生长因子对于中胚层及神经外胚层来源的细胞都有强烈的促有丝分裂作用，能有效的促进细胞增殖，自我更新，诱导间充质干细胞向韧带细胞方向分化[4-5]。并在损伤肌腱的重塑形及提高新生肌腱的生物力学强度上有着重要作用[6]。外源性碱性成纤维细胞生长因子对细胞增殖促进作用有明显的量效关系，Hankemeier等[7]发现低剂量碱性成纤维细胞生长因子(3 μg/L)添加于培养基中能使间充质干细胞增加Ⅰ、Ⅲ型胶原、平滑肌肌动蛋白、波形蛋白和纤维粘连蛋白mRNA的表达，其均为韧带的特异性细胞外基质。MacLean[8]研究发现6 μg/L是碱性成纤维细胞生长因子促进前交叉韧带纤维母细胞的增殖的最佳浓度，其可能通过调节细胞分解代谢通路来产生作用，增加或减低浓度都将对其效果产生影响。通过直接添加的方式将碱性成纤维细胞生长因子加入培养基中这种传统的方法行之有效，但在细胞培养、分化过程中，由于不断的换液、传代操作可能造成生长因子浓度有较大的波动，从而影响了最终的生物学效果。学者们发现将支架结构与外源性碱性成纤维细胞生长因子及种子细胞复合，再通过生物反应器进行机械刺激，可以更好地模拟机体的内环境，有效促进TEL生长，并且有效避免了由于频繁换液而导致的生长因子浓度丢失。Sahoo 等[9]将碱性成纤维细胞生长因子直接与蚕丝支架复合，再与间充质干细胞进行复合培养，3周后观察到间充质干细胞增殖明显，其中Ⅰ、Ⅲ胶原蛋白含量及生物力学性能明显优于未复合碱性成纤维细胞生长因子的对照组。此外，采用病毒及质粒等载体将碱性成纤维细胞生长因子基因转入种子细胞内，可使其蛋白在一定时间内稳定的表达，并发挥外源性碱性成纤维细胞生长因子的生物学效能[10-11]。Tang等[12]将含有碱性成纤维细胞生长因子基因的腺病毒注入来亨鸡趾深屈韧带断端后，发现在前8周的韧带修复时间内，碱性成纤维细胞生长因子能明显的增强修复韧带的生物力学强度，并有目标蛋白的稳定表达。虽然碱性成纤维细胞生长因子在肌腱愈合过程中促进作用明显，但学者同时也发现碱性成纤维细胞生长因子有加重了肌腱粘连的倾向[13]，其可能与鞘内肌腱细胞的胶原分泌有关，具体机制目前尚在研究中。 2.2 胰岛素样生长因子1 胰岛素样生长因子1是由70个氨基酸组成的碱性单链多肽，因其结构与胰岛素类似而得名。韧带细胞和腱鞘细胞均可表达胰岛素样生长因子1 mRNA并合成蛋白，在正常情况下与胰岛素样生长因子1特异结合蛋白结合，形成一个非活性的胰岛素样生长因子1蛋白库，组织受损后，一些酶释放解开结合的非活性胰岛素样生长因子1蛋白并激活它胰岛素样生长因子1。损伤的早期，胰岛素样生长因子1在损失周围的多种细胞基质内均能发现，其中以腱鞘外膜中浓度最高，且胰岛素样生长因子的分泌量与肌腱愈合有着明显的相关性。胰岛素样生长因子1是潜在的促有丝分裂剂，能加快韧带细胞mRNA的转录和各种蛋白质的翻译合成并加速韧带细胞有丝分裂的完成，以剂量依赖性的方式促进胶原、蛋白多糖等细胞外基质的分泌[14]。体外实验表明，外源性胰岛素样生长因子1在有效浓度范围内与肌腱细胞的增值有明显的量效关系。 10 μg/L是刺激肌腱细胞生长的最佳浓度，增加或降低浓度均可能导致细胞增值速率降低[15]。胰岛素样生长因子1在肌腱愈合过程中的促进作用显著，Provenzano等[16]通过对大鼠内侧副韧带损伤模型的观察发现，皮下注射胰岛素样生长因子1能有效提高韧带的最大负荷、极限应力、弹性模量，Ⅰ型胶原等相关蛋白的表达量明显增加。但由于其半衰期较短，体外培养时采用外源性生长因子时需反复给药，目前学者多采用转基因技术克服此缺陷。Schnabel[17]将胰岛素样生长因子1转入马间充质干细胞中，并将其运用于马的屈趾浅腱损伤模型的治疗，在伤后2，4，6，8周时分别取材并进行RT-PCR检测，结果显示Ⅰ型胶原基因显著增多，修复后肌腱中胶原含量明显增加，修复韧带生物力学强度明显增强。近年来，研究发现胰岛素样生长因子1是衡量肌腱新陈代谢重要的生物学指标[18]。表达水平与韧带的耐力适应性有显著关系，韧带疲劳损伤的早期胰岛素样生长因子1含量明显增加，而且其变化较韧带细胞发生凋亡及局部炎症反应发生要早[19]。 2.3 骨形态发生蛋白12 骨形态发生蛋白12是转化生长因子β超家族的成员，正常韧带的腱鞘内膜及血管周围组织中均可以检测到骨形态发生蛋白12。韧带损伤后，特别是损伤早期，组织中骨形态发生蛋白12的合成明显增加，研究表明骨形态发生蛋白12通过旁分泌能有效促进损伤组织周围的韧带细胞增殖，并诱导Ⅰ、Ⅲ型胶原前体的分泌[20-21]。小剂量的骨形态发生蛋白12(10 μg/L)刺激12 h能使小鼠间充质干细胞高效的表达肌腱相关蛋白(Scleraxis蛋白、腱调蛋白)，将该细胞与胶原支架复合后植入小鼠体内，3周后其肌腱相关蛋白仍明显高于对照组[22]。相比骨形态发生蛋白家族的其他成员，骨形态发生蛋白12诱导间充质干细胞向韧带细胞分化的能力较强，而向成骨细胞分化的能力较弱[23]，能有效克服间充质干细胞在体内培养过程中的异位骨化问题。Wang等[24]通过电穿孔法将骨形态发生蛋白12基因直接导入间充质干细胞间充质干细胞中，观察发现间充质干细胞可定向分化为腱细胞，分化后的细胞中可以检测到Scleraxis蛋白mRNA表达，其被认为是韧带细胞表面特异性标志物[25]，其他肌腱相关蛋白的表达也较未转染的间充质干细胞明显增加。采用基因修饰的方法将骨形态发生蛋白12用腺病毒携带后转染人间充质干细胞，21 d后细胞形态与正常韧带细胞表型类似，韧带组织的细胞外基质如Ⅰ、Ⅲ胶原、波形蛋白、弹性蛋白、韧带蛋白和腱调蛋白等的表达量较未转染组明显增加[26]。骨形态发生蛋白12还是韧带-骨界面处形成连接的重要调节因子。骨形态发生蛋白12能促进使界面处的纤维软骨形成，使局部黏多糖含量增加，从而增加韧带愈合后最大负荷强度。 2.4 转化生长因子β 人类的转化生长因子β家族有3个亚型，即转化生长因子β1 、转化生长因子β2和转化生长因子β3，三者在韧带损伤修复中扮演着不同的重要角色。在大鼠髌韧带损伤模型中应用重组人转化生长因子β1、β2、β3发现：3种亚型均可促进Ⅰ型胶原前体 mRNA含量不同程度的增加，三者均能促进肌腱组织愈合，且具有明显的协同作用[27]。其中，转化生长因子β1的体内分部最为广泛，与肌腱损伤修复过程的关系也最为密切，是目前研究的热点。转化生长因子β1是韧带平滑肌肌动蛋白α分泌重要的信号分子，激活该信号通路能使纤维母细胞向肌纤维母细胞分化[28]，从而促进损伤韧带的修复。利用转化生长因子β1转染间充质干细胞并植入兔跟腱损伤模型中，Ⅰ型胶原的分泌明显增加，其修复的韧带在生物力学性能和修复时间上明显优于对照组[29]。但多数学者认为转化生长因子β1的过表达是导致肌腱粘连愈合的主要原因。Kim[30]认为外源性的转化生长因子β1能够促进Ⅲ型胶原的分泌，加速韧带-骨损伤处的愈合，但此作用机制可能更多的与瘢痕修复相关。研究发现转化生长因子β1表达水平和腱鞘成纤维细胞、腱外膜细胞、腱内膜细胞Ⅰ型胶原的基因表达水平及周围组织的粘连有明确的相关性[31]。在体内使用中和抗体降低损伤韧带周围转化生长因子β1表达水平，可使Ⅰ型胶原的在损伤韧带周围的表达量明显减少，从而有效的缓解了韧带术后周围组织的粘连，术后韧带的活动度明显改善[32-33]。从而证明了转化生长因子β1是韧带损伤后瘢痕形成的重要调控因子。"

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