基于金纳米探针的支架材料降解与骨再生动态监测及定量分析

The basic principle in tissue engineering is to build and promote the regeneration of defective tissue with the biodegradable scaffolds, which should have a suitable biodegradation rate matching to the progress of tissue regeneration. Currently available methods for tissue and scaffold degradation analysis, such as histology and direct mechanical measurements, are not suitable for continuous monitoring of the same sample in vivo as they destroy cells, tissue matrix and scaffolds. In addition, different samples are prepared and measured at varying times, but high tissue growth deviation between specimens and the need for monitoring tissue growth and scaffold degradation at different times requires large sample numbers for statistical analysis. In order to solve this problem, we intend to develop a dual-mode imaging strategy for in vivo noninvasive trafficking of scaffold degradation and tissue regeneration, using a novel nanoprobes and imaging technology. In this project, we will prepare and modify gold cluster used as a dual-functional contrast agent/probe with favorable near-infrared fluorescence and X-ray attenuation properties. Then, the gold cluster will be grafted onto the PLGA scaffold which has no fluorescence under laser excitation and cannot be imaged by Micro CT. In this project, the bone marrow mesenchymal stem cells will be seeded onto the as-prepared gold cluster-labeled scaffolds and implanted into the rat skull defect model to evaluate this experimental strategy and the hypothesis. We will optimize the preparation technology of the gold cluster-labeled scaffolds, and investigate the biocompatibility and biodegradability in vitro/vivo. Finally, the dynamic in-situ monitoring of biodegradation rate of the gold cluster-labeled scaffolds and tissue regeneration will be further verified using a dual-mode imaging of near-infrared fluorescence and Micro CT. The research results achieved in this project will not only address the practical problems for monitoring the degradation rate of the tissue engineering scaffold and tissue regeneration progress, but also provide a novel strategy to develop new generation intelligent biodegradable scaffolds for tissue engineering.

在应用组织工程技术再生组织过程中，理想的支架材料降解速率必需与组织再生速率相匹配，而无创动态监测支架材料的降解仍然是本领域面临的难题。已有同位素、荧光染料示踪等技术存在核素污染、荧光信号不稳定甚至淬灭等问题。基于金纳米团簇的荧光和对X射线的衰减特性，我们提出假设，将金纳米探针标记材料，通过荧光和显微CT双模式活体成像技术，建立无创动态监测与定量分析支架材料体内降解与组织再生匹配的体系。预初实验利用荧光金纳米团簇标记的PLGA裸鼠皮下埋置模型，初步证实了应用金纳米探针示踪支架材料降解的可行性。本项目在此基础上将进一步探索近红外荧光金纳米探针制备技术，并以此示踪PLGA支架材料，在大鼠颅骨缺损再生修复模型中深入探讨材料降解与骨组织再生速率匹配的科学问题。该体系的建立将解决体内支架材料降解无创监测的难题，为可降解生物支架材料的设计和应用提供可靠的评估手段。

应用仿生支架模拟组织再生微环境过程中，理想的仿生支架降解速率必需与组织再生速率相匹配，而无创动态监测支架材料的降解仍然是本领域面临的难题。已有同位素、荧光染料示踪等技术存在核素污染、荧光信号不稳定甚至淬灭等问题。本项目基于金纳米团簇的荧光和对X射线的衰减特性，建立仿生支架降解过程的动态监测及定量分析技术，实现了再生微环境的动态监测，为研究组织再生过程和机制提供了工具。本项目经过四年的研究，已完成项目计划内容。项目执行期间，发表SCI论文9篇，其中影响因子（IF）>20分的SCI论文1篇，IF>10分SCI论文2篇，IF>5 SCI论文6篇。还有3篇标注本项目的SCI论文在投稿之中。申请国家发明专利3项，其中本项目核心专利已授权。招收硕士生1名，参与培养硕士、博士、博士后4余人。本项目主要研究成果和结论：（1）制备了近红外金纳米团簇，荧光和对X-射线衰减等性能稳定，无细胞毒性。（2）利用金纳米团簇标记PLGA，制备了荧光PLGA骨支架。（3）荧光和CT双模式成像监测荧光PLGA支架体外降解情况，通过相同时间点测量支架的质量，分析支架质量变化和成像结果之间的线性关系。（4）裸鼠皮下埋置模型及动态监测荧光PLGA在体内降解情况，用生物统计学方法分析支架降解过程中荧光和显微CT信号变化的线性关系。（5）大鼠颅骨再生模型及其动态监测荧光PLGA在不同时间点进行动态成像，运用统计学分析，支架材料降解过程荧光和显微CT信号变化和颅骨再生匹配情况，三者之间的相关性。（6）最后采用组织切片染色法验证荧光和Micro CT双模式成像技术监测的结果。（7）在此技术基础上，研制了一系列物理、化学以及生物微环境因素调控的骨再生微环境仿生支架，研究成果均已发表。（8）还把骨再生仿生技术和理念拓展到皮肤、肌腱等软组织再生微环境的构筑。例如，通过力学、拓扑结构、电信号等多重仿生，构建了肌腱特异性压电支架，该理念已获国自然面上项目。