仿生活性丝素蛋白-P(LLA-CL)微纳米纱线针织支架用于兔跟腱再生

There is still no idear scaffold for tendon tissue engineering, this project will develop a nanoyan tendon scaffold by electrospining which could biomimic the nanofibrous structure of Extrocellular Matrix and promote the tendon tissue regeneration. Firstly we will prepare four kinds of nanofiber yarns, that is, SF-P(LLA-CL) nanofiber yarn, PLGA-SF-P(LLA-CL) micro/nanofiber yarn, PET-SF-P(LLA-CL) micro/nanofiber yarn and growth factor loaded PLGA-SF-P(LLA-CL) micro/nanofiber yarn by electrospinning and emulsion electrospinning using the nanoyarn spinner instrument developed by our Lab. The morphology and mechanical properties of nanofiber yarn will also be investigated. The further step is to knit the four kinds of nanofiber yarns into four kinds of tendon scaffolds (knitted SF-P(LLA-CL) nanofiber yarn scaffold, knitted PLGA-SF-P(LLA-CL) micro/nanofiber yarn scaffold, knitted PET-SF-P(LLA-CL) micro/nanofiber yarn scaffold and knitted growth factor loaded PLGA-SF-P(LLA-CL) micro/nanofiber yarn). The structure morphology, mechanical properties and biodegradability of the three knitted scaffold will be tested. The tendon cells will be cultured on the three scaffolds to investigate how fast cells proliferate on different scaffolds, how cells infiltrate three dimensionally into the scaffolds, how the gene express on the scaffolds and how the growth factor influence the cell proliferation speed and gene （collagen I, collagen III, tenascin C）expression level. Growth factor (bFGF and IGF-1) release behavior from knitted growth factor loaded PLGA-SF-P(LLA-CL) micro/nanofiber yarn scaffold will also be investigated. Finally the four knitted scaffolds will be implanted into rabbit to replace the achilles tendon defect(1.5cm) to investigated the new tendon regeneration. At different implantation time the regenerated tendon will be used to test the tensile strength and H-E histology analysis. From the animal test to show that the nanoyarn structure and growth factor can promote the tendon tissue regeneration, PET can enhance the mechanical properties of regenerated tendon.

肌腱组织再生期待给出力学性能强、具有生物活性的仿生的组织工程支架，本课题利用静电纺纳米纱线制备出仿生组织细胞外基质的新型肌腱支架，并使之负载生长因子，有望促进组织的再生。首先是创制出连续纳米纱线制备设备，用此设备制备出丝素-P(LLA-CL)纳米纱线、PET纤维增强型维纳米纱线、PLGA纤维增强型维纳米纱线和乳液静电纺含生长因子微纳米纱线，检测纳米纱线的形态结构和力学性能。然后将此四种纱线针织成四种纳米纱肌腱支架，表征支架的形态结构、测试力学性能和体外可降解性，研究生长因子从支架中的缓释速率。将肌腱细胞在支架上培养，研究细胞在针织支架上的三维长入情况，及生长因子和支架结构对细胞增殖速率和肌腱相关基因表达的影响。最后将四种支架植入兔子的跟腱缺损（1.5cm），不同时间取出再生肌腱测定其力学强度和组织学形态，观察支架材料降解过程和肌腱组织再生过程，揭示纳米纱结构及生长因子对肌腱再生的促进作用。

现有合成高分子聚合物制成的单一组分支架缺乏生物活性，不足以引导肌腱/韧带组织生长。为了克服这种限制，本研究制备由“皮芯”结构纳米纤维纱线制成的针织复合支架，用于引导肌腱组织再生。通过纳米纱线制造设备将聚己内酯（PCL）或丝素蛋白（SF）/聚乳酸-己内酯（PLCL）纳米纤维包裹在PCL微米纤维周围，产生皮芯结构纳米纤维纱线（PCL-PCL和PCL-SF/PLCL纱线），并将纱线针织成三维支架用于肌腱修复。PCL-SF/PLCL支架可以促进肌腱组织再生和重塑，并且再生肌腱具有较好的结构和生物力学性能。.为了解决肌腱/韧带组织工程支架在骨隧道段缺乏骨传导性的问题，同时模拟肌腱-骨界面四个不同区域的过渡结构。本研究利用静电纺丝技术制备四种负载不同含量的羟基磷灰石（HA）纳米纤维纱线，然后利用编织方法将纱线按照梯度顺序组装成HA梯度支架。体外生物学结果显示，支架对BMSCs和小鼠胚胎成骨细胞前体细胞（MC3T3-E1）均表现出良好的细胞相容性。支架的矿化区段促进了MC3T3-E1的增殖，并且诱导BMSCs的成骨分化；而非矿化区段促进BMSCs增殖和成肌腱细胞分化。HA梯度支架可以在空间上调控BMSCs的分化表型，对促进肌腱-骨界面组织的再生和修复有潜在作用。.为了进一步解决人工韧带在骨隧道内难以与骨融合，植入后造成骨隧道扩大的突出问题。本研究结合静电纺丝、纺织技术以及热致相分离（TIPS）方法设计并制备出一种三相结构的骨-韧带-骨一体化支架，其中负载结缔组织生长因子（CTGF）的纳米纤维纱线编织支架作为一体化支架的韧带部分（中间位置），负载介孔羟基磷灰石（MHA）和去铁胺（DFO）的聚乳酸（PLA）热致相分离支架作为一体化支架的骨隧道部分（两端位置）。研究发现，负载DFO@MHA纳米颗粒的PLA支架促进了骨组织工程中的血管化，加速骨隧道修复。SF/CTGF@PLCL支架组的再生韧带组织表现最好，显示出类似于天然韧带的结构，具有高度卷曲胶原纤维束的特征。