生物活性一体化层状支架修复关节软骨缺损及促界面整合机制的研究

The poor results for repairing articluar cartilage lesions with relatively large sizes and the deterioration of regenerated articluar cartilage still remain a key challenge in the field of articluar cartilage repair. This project aims to build a new type of integrated scaffold/cell complex that has interfacial bioactivity and stratiform structures in order to repair the articular cartilage defects with relatively large sizes and extend the long-term effects of restoration. A RGD-modified dextran-poly(lactide) (R-Dex-PLA) copolymer with optimized compositions and structures is first synthesized. R-Dex-PLA is then blended with some other materials, such as chitosan, collagen, silk fibroin and nano-hydroxyapaptite to produce various composites for the fabrication of scaffolds. The stratiform scaffold is designed in such a way that it contains typical three layers that can mimic the composition and structures of articular cartilage: (1) a cartilage-like layer with gradient compositions and structures; (2) a biomimetic calcified layer built by electrospun nano-fiber membranes; and (3) a bone-like layer with gradient mechanical modulus. All these layers are integrated together to form into a full scaffold via a three-dimensional printing technique, and the resulting scaffolds are endowed with designated compositions and porous structures. In addition, two types of microspheres loaded with different growth factors are further fixed on the bottom surface corresponding to the bone-like layer whilst some cyclic peptide gels having certain in situ induced functions are attached to the side surface of the full scaffold. After being seeded with two types of cells in the separate layers and cultured in a dual-chamber co-culture system, the integrated scaffold/cell complex with interfacial bioactivity and layered structures are thus achieved. The so-produced complex will have some abilities to help reconstruct articular cartilage and improve the interface integration between neocartilage and host-tissue by way of its high biomimetic characteristics and interfacial bioactivity, and as a result, the improved repair quality and enhanced long-term function maintenance for the neocartilage could be achieved. The presently proposed solutions will not only help to deeply understand the micro-mechanisms by which the scaffold/cell complex together with growth factors can synergistically regulate the structures and properties of newly regenerated tissue and enhance the effective intergation between guest-tissue and host-tissue but also possibly develop a new method or technique for articluar cartilage repair in the clinical treatment.

较大直径关节软骨缺损修复效果差和后期退化是亟待解决的难题。本项目提出了新的解决方法。将合成得到的RGD短肽修饰葡聚糖-聚丙交酯共聚物与壳聚糖、胶原、丝素蛋白和纳米羟基磷灰石等材料进行多种复合经电纺丝和三维打印方法组装成一体化层状支架。支架由梯度成分和结构的仿非钙化软骨层、纳米纤维膜搭建的仿钙化层和梯度力学模量仿骨层构成。另分别将载血小板源性生长因子和骨形态发生蛋白的两类微球加载到支架仿骨层底面，将载c(RGDfK)环肽胶体加载到支架侧面。复合两种细胞后经双室细胞共培养获得具有生物活性的支架/细胞复合体。该复合体将通过其精细可控组成和结构及活性协同构建具有类似正常结构的新生关节软骨并使之与宿主有效整合从而将显著提高关节软骨缺损修复质量和防止后期退化。本项目不仅有望揭示支架、细胞和活性生物分子协同调控新生关节软骨与宿主整合的微观机制，还可能为较大直径关节软骨缺损修复提供新的治疗方法。

合成葡聚糖-聚丙交酯(Dex-PLA)共聚物用于制备可打印增强型水凝胶的主要组分。将两种活性因子分别加载到海藻酸钠和明胶微球，再将这些微球与水凝胶复合建立微球/凝胶体系。该微球/凝胶体系具有可打印成型性并可有效控制因子释放和保持两种因子的活性。用共沉淀方法合成制备壳聚糖(CH)/纳米羟基磷灰石(HA)复合物, 再将所获得CH/HA复合物与丝素(SF)进行混合，经静电纺丝方法制备得到了4种不同组成比的纳米纤维膜。按CH、SF和HA三种组分形成各自组成梯度的方式将4种纳米纤维膜分层连续电纺而加工成梯度仿生整体膜。将该仿生膜用作层状支架的仿钙化层。将软骨细胞加载于层状支架的仿软骨层同时将成骨细胞加载于层状支架的仿骨层，经体外双室细胞培养证实这类支架能有效支持透明软骨细胞和成骨细胞生长并维持各自表型。基于该型层状仿生支架的组成和加工条件, 制备包含Dex-PLA/CH/II型胶原仿软骨层、纳米纤维仿钙化层和Dex-PLA/HA/CH/I型胶原仿骨层的层状支架。将双因子微球/凝胶加载到层状支架仿骨层底部，将含有活性环肽凝胶加载到支架侧面, 所获得支架用于动物体内关节软骨缺损修复。结果证实仿生层状支架能显著促进新生组织形成透明软骨并增强新生软骨与宿主组织的整合。仿生层状支架结合生长因子和活性多肽能明显提高关节软骨缺损的修复质量。研究结果也部分阐明了这类支架修复关节软骨缺损的整合机理, 为后续应用转化研究打下了良好基础。