基于原位软骨成像及3D打印技术构建仿生支架修复软骨缺损的研究

The repairing of articular cartilage injury is a major challenge in the field of sports medicine. At present, the clinical repair treatments can only offer some alleviation of symptoms. Recently, tissue engineering and 3D printing technology extend and promote the methods and materials of cartilage repairing. Clinical 3D printing technique is mostly based on image data, like CT (Computed Tomography) or MRI (Magnetic Resonance Imaging). However, cartilage 3D printing materials, which is usually molded by computer software, cannot simulate the in situ microstructure of cartilage. In recent years, the development of two-photon laser scanning confocal microscopy for biomedicine has facilitated the dynamic noninvasive imaging and measurement of many living tissues, like skin, liver, nerve or muscle, but articular cartilage. Therefore, in this study, we will use a laser confocal microscope to excite and collect the two-harmonic imaging of cartilage matrix, which will be used to construct cartilage in situ micromorphology and evaluate the overall damage and repairing of articular cartilage. And this in situ cartilage imaging will be verified and quantified with an animal model. In addition, according to the micromorphology of cartilage, we will build a 3D printing mold for the construction of biomimetic cartilage scaffolds, which will be test the biocompatibility and ability of cartilage repairing in vitro and in vivo. Finally, using epigenetic research methods, we will explore the key regulatory mechanisms of the cartilage repairing of this biomimetic 3D-printing scaffold, which may promote further functionalization of cartilage repairing materials.

关节软骨损伤修复是骨科运动医学领域的重大挑战，临床上的治疗方法仍不能提供理想的修复效果。组织工程及3D打印技术的发展为软骨修复提供了新的方案。目前，临床3D打印多是基于影像学资料模拟特定组织的原有形态构建的，关节软骨结构微观、成像复杂，难以提供用于3D打印的图像数据，而无法模拟构建软骨的原位微观形态。近年来，双光子激光扫描共聚焦显微镜在生物医学领域的应用，使活体无创性动态成像及微观测量成为现实。但是，关节软骨微观结构的原位动态成像还没有实现。本项目拟利用激光共聚焦显微镜激发并采集软骨组织的二次谐波动态成像，在动物模型上验证原位软骨成像技术，并建立原位软骨成像的定量评估体系；进一步利用原位成像技术获取软骨微观形态，构建3D模具，模拟打印仿生支架，并在体内外验证支架的生物相容性及软骨修复能力；最后，初步探索仿生支架促进软骨修复的关键调控机制，为后续生物支架的结构及功能优化提供参考。

关节软骨损伤修复一直是运动医学研究与临床治疗的棘手问题。组织工程及3D打印技术的发展为软骨修复提供了新的方案。目前，临床3D打印多是基于影像学资料模拟特定组织的原有形态构建的，而关节软骨成像复杂，难以提供用于3D打印的图像数据，无法模拟软骨的原位微观形态。.基于此背景，本项目利用关节软骨二次谐波成像原理，建立双光子激光扫描共聚焦显微镜原位软骨微观形态观测方法，结果显示原位软骨成像方法具有组织特异性，可以精准显示软骨组织，且不需进行其他染色处理，并与传统甲苯胺蓝染色对比，结果显示原位成像荧光强度与损伤程度相关，验证原位成像定性定量测量效果。.在关节软骨原位成像基础上，利用计算机模拟软骨原有的微观结构，将全层（最深可达500微米）XYZ三轴扫描转换成3D打印模型，构建光固化聚己内酯树脂（EFL-PCLMA-3080）打印软骨仿生支架。在课题实施过程中，项目组发现软骨仿生支架植入后可达到软骨修复应力要求，但深层软骨下骨力学强度大，需构建生物力学强度大的骨诱导材料，才能在修复过程中为表层软骨修复提供力学支撑。为此，我们采用激光粉床融合Laser-powder bed fusion（L-PBF）制备拓扑有序多孔锌粉，进行铺粉、粉末熔化和固化（粉末层厚度为60μm，能量密度为39.0J/mm3），最后切割除粉制备出多孔可降解纯锌圆柱体，填充深层软骨下骨缺损，借助其力学特性及降解的锌离子促进骨愈合的特性，加强其对上层软骨修复层的支持，在巴马香猪大动物实验中验证骨软骨缺损填充、表层软骨修复效果，并对修复组织进行相关基因检测，仿生支架修复组织内成软骨基因表达上调，提示仿生结构的生物支架更有利于软骨基质的形成。项目组还研发了生物材料修复软骨损伤的植入器械及操作流程。.本项目使双光子激光扫描共聚焦显微镜在关节软骨无创性原位成像及微观测量成为现实，该方法简便快捷，不需要额外染色处理，具备推广应用空间。另外，基于组织原位微观结构构建仿生支架的实施及概念引入，为组织工程生物材料研究提供了新思路。