动态力学刺激对纤维环-髓核组织工程化构建的影响

Tissue engineering provides a revolutionary opportunity to restore function to degenerated intervertebal disc (IVD), however, there are many challenges facing the construction of tissue-engineered IVD, especially the effects of mechanical stimulation on the construction need further study. In our prior study, integrative scaffolds of anulus fibrosus-nucleus pulposus (AF-NP) have been fabricated with a novel combining technique, the mechanical stresses of IVD were mimicked with three-dimensional finite element analysis. In this project, new bioreactors will be developed for IVD culture, that could provide bionic dynamic mechanical loads including compression and torsion. Integrative AF-NP composites will be engineered with AF and NP cells, as well as IVD-like cells induced from adipose-derived stem cells. The engineered composites will be cultured in bioreactor with bionic dynamic compression and/or torsion loading. Then the effects of dynamic mechanical stimulation on the construction of AF-NP composites can be studied, including cell proliferation, apoptosis, distribution, differentiation and gene expression, extracellular matrix secretion and arrangement, tissue reconstruction and mechanical properties in different portion of the composites. The effects of mechanical stimulation on the integrative fusion of AF and NP at their junction will also be studied. So the rule of mechanical modulation for the construction of IVD can be explored. As far as we know, the effects of dynamic mechanical stimulation on the construction of tissue-engineered IVD has not been reported in the literature. This project will no only improve the construction technique of tissue-engineered IVD, but also reveal the relation between structure and mechanical properties of IVD, that will lay foundation for the reparation and reconstruction of IVD.

组织工程有望为椎间盘疾病的治疗提供理想的重建措施，但目前构建组织工程椎间盘存在很多挑战，其中力学环境对椎间盘构建的影响亟需深入研究。前期研究中我们研制了纤维环-髓核双相一体化支架，通过有限元技术对椎间盘应力状态进行了模拟仿真研究。本课题研制用于椎间盘培养的生物反应器，可施加动态压缩、扭转等仿生力学载荷；利用纤维环细胞和髓核细胞，以及向椎间盘样细胞诱导分化的脂肪干细胞分别构建纤维环-髓核复合组织，采用生物反应器在动态压缩和/或扭转载荷下培养；研究动态力学刺激对纤维环-髓核一体化构建的影响，包括不同部位的细胞增殖、凋亡、分布、分化及基因表达，细胞外基质分泌及排列，组织结构优化及力学特性，特别是力学刺激对纤维环-髓核交界区融合生长的影响，探索椎间盘构建的力学调控规律。此研究国内外未见报道，研究成果将从源头上提高椎间盘构建技术，并能揭示椎间盘结构-力学功能的关系，为椎间盘的修复与重建提供理论基础。

椎间盘疾病导致的腰腿疼严重影响患者的生活和工作，组织工程有望为椎间盘疾病的治疗提供理想的重建措施，但目前构建仿生组织工程椎间盘存在很多挑战，其中力学环境对椎间盘构建的影响亟需深入研究。由此进行了三方面的研究。. 第一，在组织工程椎间盘的构建方面进行了探索，以期获得理想的组织工程椎间盘。使用共培养的方法成功将脂肪来源的干细胞向软骨细胞分化。以骨基质明胶分别和脱细胞髓核基质、脱细胞软骨基质构建了纤维环-髓核一体化复合双相支架。以丝素蛋白(Silk Fibroin, SF)分别构建了纤维环支架和髓核支架，并构建了纤维环-髓核一体化复合双相支架。PKH26 标记结合活体荧光成像技术在组织工程椎间盘的应用，可以追踪细胞在体内的生物活性。以聚己内酯[poly(caprolactone), PCL]为材料制备了环形取向PCL纤维环支架。进一步制备了PCL/SF（1:1）纤维膜，并组装成仿生斜交叠层结构，能够引导纤维环细胞取向性生长。. 第二，生物反应器改进方面。以前期制作的力学加载装置为基础，研制出动态压缩耦合扭转载荷的生物反应器，可同时实现在液体培养环境下的压缩或/和扭转载荷加载，从而为椎间盘组织的培养提供了接近于生物体的液体环境和力学环境。. 第三，动态力学刺激对组织工程椎间盘构建的影响方面。使用生物反应器对髓核细胞-丝素蛋白多孔支架复合体施加动态力学刺激后，结果显示10%应变组的蛋白多糖和II型胶原含量较5%和0应变组明显增加；10%和15%应变组细胞的增殖活性较5%和0应变组明显增高。利用生物反应器对环形取向SF支架-纤维环细胞复合体行动态压缩力学刺激，结果表明适宜的力学刺激是纤维环细胞增殖和分泌的有利因素，且在1Hz频率，Ⅰ、Ⅱ型胶原在最佳压缩幅度10%，GAG和压缩弹性模量最佳压缩幅度15%，DNA最佳压缩幅度5%。适宜的力学刺激有利于椎间盘细胞的生长和细胞外基质的分泌、排列，有利于纤维环和髓核交界面的融合，初步揭示了椎间盘结构-力学功能的关系，为进一步的研究复杂动态力学刺激下对一体化功能椎间盘的构建研究打下了坚实基础。