3D打印多级有序仿生支架促进腱骨愈合的实验研究

It is a great challenge to repair tendon (ligament) with satisfactory long-term efficacy in clinic. One of the critical issues is the poor outcome of graft-to-bone interface healing, which is hard to be biologically integrated. The development of tissue engineering scaffolds provides a new aspect to solve this problem. However, the general tendon (ligament) tissue engineering scaffolds have several disadvantages such as poor control of structure, low mechanical strength, and insufficient osteogenic performance. Therefore, in this project, we propose a novel tendon-bone tissue engineering scaffold with controllable structure, high mechanical strength, and excellent osteogenic performance by using 3D printing technology. In terms of composition, calcium silicate nanowires with excellent osteogenic activity are combined with alginate which possesses the tenogenic potential. In terms of structure, the orderly arrangement of calcium silicate nanowires in the composite scaffold can be achieved by shear stress from 3D printing header. Also, the micron-oriented fibers of alginate can be constructed by "directional drying method". Thus, the composite scaffolds with "nano-micron-macro" hierarchical architectures are fabricated. The cell study and animal experiments are to explore the influence of the composition/structure dual regulatory scaffolds on tendon-bone healing, as well as the related mechanisms. Our project would provide a theoretical and experimental basis for the design and preparation of new tendon (ligament) tissue engineering scaffolds.

肌腱（韧带）重建手术长期疗效不佳是临床亟待解决的难题，其中一个关键问题就在于腱骨愈合不理想，移植物与骨无法形成生物学融合。组织工程支架的发展为解决这一难题提供了新的手段，但目前的肌腱（韧带）组织工程支架普遍存在结构控制差、力学强度低、成骨性能不足等缺点。为此，本课题拟采用3D打印技术构建结构精确可控、力学强度高且成骨性能优异的腱骨组织工程支架。组成上，我们将成骨活性优异的硅酸钙纳米线与具有成腱潜力的海藻酸结合；结构上，巧妙地利用3D打印针头的剪切应力实现硅酸钙纳米线在复合支架中的有序排列，并通过“定向干燥法”构建海藻酸微米取向纤维，从而制备出“纳米-微米-毫米”多级有序复合仿生支架。进一步通过体外细胞实验和体内动物实验探究组成/结构双调控支架对腱骨愈合的影响及其相关机制，为设计和制备新型肌腱（韧带）组织工程支架提供理论依据和实验基础。

腱骨缺损修复是临床上的一大难题，其原因在于损伤部位同时涉及骨和肌腱两种完全不同的组织，而普通的骨修复材料或者肌腱修复材料并不能同时满足”腱骨“修复的需求。因此，急需开发同时具备成骨和成肌腱活性的新型腱骨修复材料。本项目受天然肌腱的多级取向结构和天然骨的有机/无机复合结构的启发，通过纳米合成技术、3D打印技术以及定向干燥技术的有机组合，构建了具有”纳米-微米-毫米-厘米“多尺度取向结构的海藻酸钠/硅酸钙纳米线（Alg/CS）复合支架，系统研究了制备工艺对支架取向结构及理化性能的影响。结果表明：打印针头越小，CS取向排列越明显。虽然适量的CS复合（5-20 wt%）有利于3D打印的进行，但复合浓度越高，其取向排列越差。相反，支架拉伸强度、离子释放速率随复合增大而增大。定向干燥会增强支架的力学强度，但不影响其离子释放规律。体外细胞实验则表明：复合了适量CS（ 10 wt%）的多级取向结构支架比单独取向结构或单独复合支架有更好的细胞调控作用，包括刺激骨髓间充质干细胞增殖、排列和朝成骨分化以及刺激肌腱干细胞增殖、排列和朝成肌腱分化。体内动物模型（兔子跟腱缺损）则进一步证明了具有多级取向结构的Alg/CS复合支架能够显著地促进缺损部位的骨和肌腱再生，加速腱骨融合。本项目充分证明了通过合理的组成和结构双调控，有望协同增强支架的再生活性。而基于这一策略，项目组还构建了针对其他临床应用场景的组成/结构双调控支架，并对其普适性进行了充分验证。该项目的实施不仅为腱骨缺损修复提供了新材料，还为设计和构建新型组织再生材料提供了新思路。