Chinese Journal of Tissue Engineering Research ›› 2017, Vol. 21 ›› Issue (18): 2940-2945.doi: 10.3969/j.issn.2095-4344.2017.18.024
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Feng Peng-fei1, Wang Ji-hong2, Ji Yun-tao2, Zhao Jia-li2
Received:
2017-02-14
Online:
2017-06-28
Published:
2017-07-07
Contact:
Wang Ji-hong, Chief physician, Department of Hand and Foot Microsurgery, Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
About author:
Feng Peng-fei, Studying for master’s degree, Physician, Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
Supported by:
the Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2016MS0822; the Young Talent Team Project of the Inner Mongolia Medical University in 2015, No. NYTD-2015108; the Science and Technology Plan of Inner Mongolia Autonomous Region in China
CLC Number:
Feng Peng-fei, Wang Ji-hong, Ji Yun-tao, Zhao Jia-li. Clinical advances of tendon tissue engineering materials in tendon injury[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(18): 2940-2945.
2.1 支架材料及制备研究 寻找一个有效的肌腱组织工程支架已受到显著关注,包括天然材料、人工材料。一些研究对天然和人工的混合材料已进行了研究,相信它会促进细胞的生长,同时提供机械支持的肌腱重塑[3-4]。除了支架材料的性能例如支架长度,以及其经历机械性刺激是否影响修复肌腱的极限强度[4]。跟腱修复最佳的材料可使跟腱缺损处自然和快速桥接,并且在8周内形成富含胶原蛋白组织的有条理的完整编织材料[5]。另一个支架的重要特征可能是可释放趋化分子,在降解的基础上促进祖细胞的招募[6]。 支架材料的研究和开发是组织工程的关键所在,为细胞生长提供了稳定的外部环境,如果没有合适的支架来供给肌腱细胞最适合的生存环境,种子细胞就会流失、死亡[7]。制备良好生物相容性、力学性能的三维仿生支架材料是组织工程肌腱在临床肌腱损伤再生修复治疗中急需解决的问题[8]。理想的肌腱组织工程支架应该具有适度的机械性能、良好的生物活性、良好的生物相容性、与正常胶原组织相匹配的力学强度、吸水率及三维立体结构等[9]。 目前常用的有天然高分子材料、生物衍生材料、人工合成材料及复合材料等。其中天然高分子材料主要有蚕丝、小肠黏膜下层、胶原、衍生肌腱支架材料等,保留了组织正常的三维网架结构,组织相容性好,但力学性能较差、降解速度快[10]。人工合成高分子材料主要为聚乳酸和聚羟基乙酸、聚乳酸-聚羟基乙酸共聚物、聚磷酸钙纤维等[11],它们有良好的力学性能和降解性,但存在亲水性低、细胞黏附性能差的缺点[11-12]。复合材料作为以上两者的有效结合,在临床应用中具有一定的潜 力[13]。生物衍生材料取自于生物体内,是由天然生物组织经过加工之后而形成的一类无生命活力的材料。生物衍生材料最大的优势在于经过适当的处理,其最接近人体的网架结构、生物力学性能,还具有正常的生理活性和功能等,是未来生物医学材料发展的方向之一。 2.1.1 天然高分子材料 天然高分子材料是广泛存在于动植物体中或可从自然环境中直接得到的一类天然活性高分子,也是人们最早使用的生物材料之一[14],具有良好的生物相容性、自身及其降解产物无毒、不易引起免疫排斥反应、保留了组织正常的三维网架结构、降解速度可调性大、植入人体后无刺激性及可被人体吸收等优点[15]。目前报道的天然高分子材料有从桑蚕体内分泌的蚕丝经再生制得的丝素纤维与丝素膜,还有脱细胞真皮基质、壳聚糖、血纤溶蛋白、透明质酸、海藻酸盐、胶原/明胶等[16]。 现在脱细胞肌腱组织经常作为支架的选择,这种类型的支架是自然衍生的,它保留了原生的超微结构、生化成分和肌腱细胞外基质(细胞外基质)的抗张强度。此外,在天然肌腱(细胞外基质)中发现93%的蛋白聚糖和生长因子。这些支架可通过反复冻融和核酸酶处理肌腱制备。在体外,脱细胞肌腱切片能够促进NIH-3T3成纤维细胞的增殖和依附[39]。观察在体内脱细胞肌腱-骨移植,Farnebo等[40]注意到在大鼠跟腱模型中,与未处理的肌腱-骨移植相比,脱细胞肌腱-骨移植可增加机械性能,减少免疫反应。脱细胞猪肌腱也可用于人类肌腱细胞的支架[41]。 现在已进行了人类脱细胞组织在肌腱愈合中的应用研究。其中脱细胞人异体皮肤的应用,如Graftjacket®(Wright医疗技术有限公司,阿灵顿,TN,美国)在尸体跟腱修复可明显改善机械强度和刚度[42]。在进一步的回顾性研究中表明,用GraftJacket® 治疗肌腱断裂患者有一个理想的功能恢复时间,同时没有发生并发症[43-44]。然而,这只是在少数患者中进行的回顾性研究,缺乏后期跟腱再断裂率和功能恢复时间的可靠数据。 同时也对来源于其他动物的生物材料肌腱支架进行了测试。在体外,除了缝合修复,异种移植物(Xenograft)支架与单独的缝合修复相比已显示了增强肌腱修复[45]。猪小肠黏膜下层已被用于临床作为在许多身体系统中组织改建的支架材料。临床前动物研究表明,来自于猪小肠黏膜下层的细胞外基质能够改造成肌腱组织[17]。猪小肠黏膜下层保留一些具有生物活性的生长因子,如血管内皮生长因子、转化生长因子β和成纤维细胞生长因子(FGF),可能有助于细胞在支架上的增殖和迁移[46]。动物研究表明,在移植后1个月,猪小肠黏膜下层发生约60%质量的快速降解,并在术后3个月完全吸收,到了第90天时,根据细胞结构、血管分布和组织密度的分析,由致密的胶原组织形成的细胞外基质与自然肌腱非常相似[47]。猪小肠黏膜下层的另一个优势是招募骨髓来源细胞群落,参与长期的重塑过程[46]。Suckow等[48]表明在肌腱修复中,作为支架材料的猪肾脏胶囊基质可能会产生相似的结果。 天然高分子材料的缺点是来源不同,结构与性能存在差异,且运用不同的处理方式也会造成天然高分子材料产品差异,资源有限,大批量制备困难,机械强度较低,无法人工调节且难以加工塑形等[18]。 2.1.2 生物衍生材料 生物衍生材料来自于生物体内,是将天然生物组织加工之后而形成的一类无生命活力的材料,其最大的优势在于经过适当的处理后,能具备最接近人体的生物力学性能、正常结构,还保持正常的生理活性和功能等,是未来生物医学材料发展的方向之一。在动物模型实验中,相关研究报道称此类材料已取得了显著效果[19]。周悦婷等[20]曾以生物衍生肌腱材料构建组织工程肌腱,结果显示肌腱细胞能继续增殖,细胞形态随着时间的延长而逐渐趋于正常,形成的肌腱呈白色且致密、有光泽,组织学可见胶原纤维排列较为规则,12周时肌腱细胞仍成活并分泌胶原。说明种子细胞复合生物衍生材料构建的组织工程肌腱移植后能够再生出肌腱样组织,此支架有天然肌腱的形态,生物力学接近天然肌腱,是理想的组织工程衍生肌腱支架材料。 2.1.3 人工合成材料 聚乳酸和聚羟基乙酸是目前组织工程肌腱中应用较多的的合成材料。与天然材料相比,人工合成材料有丰富的原料来源,且结构、性能等可进行任意的修饰和调控[21]。然而,它们普遍存在亲水性差,细胞黏附力弱,生物相容性不如天然高分子材料优越,在强免疫的大动物体内应用时,其降解产物可能出现机体局部大量炎细胞浸润、异物排斥等问题[22]。以上这些问题可通过复合材料的原理和方法加以解决,有人将天然高分子材料与人工合成材料互相组合[23],它也是未来修复材料研究的一个发展方向,克服了以上两种材料的不足之处,以期构造出能够符合要求的新型复合材料。Deng等[24]实验发现用单纯聚羟基乙酸作支架,因聚羟基乙酸降解快,新生胶原沿肌腱长度方向并不能形成较致密的结构,肌腱构建效果多不满意,因而应用很受限。而Yin等[25]用纳米纤维作为支架材料诱导人肌腱干细胞分化成腱时发现,排列规整有序的纳米纤维不仅可促进肌腱干细胞向肌腱细胞分化,还可阻碍干细胞分化成骨,为人工合成高分子材料应用于肌腱组织工程提供了新的活力。 现已对人工合成的可降解聚磷酸钙纤维材料进行了研究,包括其组织相容性、编织支架的物理性能、体外共同培养对细胞分化的影响、构建生物活性肌腱组织工程和修复肌腱缺损等实验,研究结果表明聚磷酸钙纤维的纤维状结构、细胞培养所需的组织相容性、物理性状及在体内可降解性良好;其缺点为pH值升高、体外降解产物堆积及表面结构不佳,但此缺点可通过体内植入及表面生物化修饰而得到优化,聚磷酸钙纤维作为肌腱组织工程支架材料、特别是在构建复合材料的肌腱组织工程支架材料研究方面有其独特的优越性及可行性[26]。 2.2 支架的设计 为了使制造出来的肌腱组织有更好的机械性能,支架设计也是至关重要的。在骨科领域,有限元的应用越来越广泛,基于化整为零,集零为整的基本思想,能够对许多结构进行精确的力学分析。将支架与有限元分析结合起来,通过建立支架材料的三维模型,用有限元数值方法来计算、模拟该材料的受力情 况[27],找出最佳应力范围,既可使细胞在支架上沿受力方向定向生长又可避免超出肌腱的受力范围[28-29]。与支架的材料相比,应力刺激不但对支架结构强度有很大影响[29],而且可以增强种子细胞的活性与细胞外基质的合成[29-31]。通过对支架进行周期性拉力或持续性拉力不同方式的试验,才能形成具有和正常人体肌腱组织接近的力学性能的组织。Legerlotz等[32]通过实验获得,不同的应力幅度和时间作用于体外肌腱细胞,其合成和分解代谢的主次不同。雷星等[33]通过对比组织工程肌腱在持续动态拉力刺激和静态非受力状况发现,前者排列逐渐有序、规则,胶原基质不断沉积,细胞/基质比例逐渐增加,而后者肌腱胶原基质缓慢降解,结构渐无序、松散,形成紊乱的极简结构。 2.3 支架的结构 研究者将兔间充质干细胞接种于不同长度的胶原凝胶支架上修复自体髌腱缺损,比较接种14 d后支架长短对构造肌腱的弹性、韧度的影响,发现长支架比短支架更优越[34]。随后,研究者采用三因素两水平的方法在体外分别研究了支架材料种类、机械刺激及长短对肌腱分化的影响,发现长的受机械刺激的胶原海绵支架比短的未受刺激的胶原蛋白凝胶支架具有更好的机械强度,胞内胶原蛋白的合成量也较高,胶原纤维排列也更紧密[35]。这说明构造支架的长短影响着组织工程肌腱的功能构建。 在肌腱构建中,编织方向有较好的力学强度,并且细胞之间孔隙适宜,在结构上纤维状支架具有多轴纤维方向,是一种良好的支架结构[36]。Ren等[12]的研究表明,排列整齐有序的纳米纤维支架比紊乱无序的结构更易促进肌腱干细胞向肌腱细胞分化,同时还能阻碍干细胞分化成骨。Qian等[37]将蚕丝编织成人工肌腱,植入兔跟腱缺损处,发现蚕丝具有良好的生物相容性,在植入一定时间内仍可保持较好的力学强度。Franklin等[38]研究发现在培养厚层细胞密集型工程组织时,接种于支架材料的种子细胞很难迁移至支架深层,或已爬入支架深层的细胞随体外培养时间的延长容易失去增殖和分泌胞外基质的能力,最后多因营养供应差而死亡。因此,在构建肌腱组织时,亦需考虑支架厚度和营养供应状况。 2.4 支架+干细胞 为了优化生物疗法的好处,把他们结合起来应用或许是有利的。单独的人类胚胎干细胞对于肌腱再生是不够的。然而,当种植到编制的胶原丝支架上时,人类胚胎干细胞可分化成肌腱细胞,并通过改变环境促进肌腱再生愈合。人类胚胎干细胞改变环境的能力通过分泌各种细胞因子,细胞外基质,和生长因子发生[49]。 支架必须由可持续提供干细胞,直到跟腱愈合的材料制成。传统的手术网能够提供间充质干细胞到损伤的跟腱处,并在大鼠模型中显示在早期可加快功能恢 复[50]。当用生物材料处理支架后,脱细胞肌腱样基质效果更优于骨样或皮肤样基质,因为干细胞更容易分化为腱细胞谱系,并具有抑制成骨细胞分化的作用[51]。 除了支架材料,还有其他的因素影响干细胞的能力。例如,如果细胞与胶原蛋白的比例过高,细胞会由于周围牵引力过大而被破坏[52]。寻找最佳的细胞与胶原蛋白之比,可改善干细胞向肌腱损伤处的持续提供。除了细胞外基质成分的浓度,精确的空间分子排列影响骨髓间充质干细胞的输送。胶原纤维的空间布局可通过角度进行调整,肌腱样支架被雕刻在某个角度。肌腱的纵向切割可促进种子细胞的形态变化和分化[53]。 由Pietschmann等[54]进行的研究显示,种植间充质干细胞的支架与满载肌腱细胞的支架相比效率更低。修复后16周,与用间充质干细胞处理的支架或者对照组相比,用种植肌腱细胞支架治疗的大鼠跟腱有更大的失效张力和横截面的比值比。 2.5 支架+生物因子 支架可通过添加外源性增强因素,添加外源基质细胞衍生因子1α到胶原丝支架将提高肌腱的生物力学性能,增加成纤维细胞样细胞的募集,减少炎性细胞的聚集,增加大鼠模型中基质细胞衍生因子1α和内源性肌腱细胞外基质的生产加工。支架也可通过添加富血小板纤维蛋白基质到断裂跟腱的两个断端之间的空白处,由支架支撑来增强。富血小板纤维蛋白基质来自离心自体血,其好处有增强生长因子、细胞因子、凝血因子的作用[55]。"
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