基于纳米级仿ECM多功能微球载体支架的组织工程个性化修复颅颌面临界骨缺损的临床前研究

Individualized reconstruction of critical size defect (CSD) of craniofacial bone is a clinical challenge. It is confirmed that bone regeneration technology based on tissue engineering is a new method for CSD reconstruction. However, there are many problems, such as the selection of scaffold materials, tissue culture in vitro, and the construction of special three-dimensional shape to adapt to different types of defects. It is reported that the extracellular matrix (ECM) has positive effects in stem cell colonization and differentiation; three dimensional culture and bottom-up technique can simulate the micro-environment in vivo to construct tissue engineering complex; Biomimetic mineralization can be used to construct inorganic nanostructures similar to natural bones; Fluorescence and magnetic properties have tracer and microsphere accumulation function; 3D printing technology can provide individualized reconstruction scheme for CSD with different defects. Thus, we propose a new systematic solution, using hydroxyapatite-chitosan microspheres of imitation ECM structure, fluorescence, magnetic and bone induced activity as multi-functional nanospheres carrier, using three dimensional culture of BMSCs/biological scaffold material and bottom-up technique to construct tissue engineering bone micro tissue, applying 3D printing technology to make personalized CSD mold. Finally, the tissue engineering bone complex with three-dimensional morphology and individual characteristics was prepared. To further complete the preclinical study, the feasibility of the system protocol was validated by nude mice-canine-primate experiments. This project will lay the foundation for the clinical application of tissue engineering technology in CSD individualized therapy.

颅颌面临界骨缺损(CSD)个性化修复重建是临床面临的挑战。研究证实基于组织工程的骨再生技术是CSD修复的新途径，但该技术存在支架选择、组织体外培养及特殊三维形态制备等难题。文献报道细胞外基质(ECM)具有促进干细胞定植和分化作用；仿生矿化可以构筑类似天然骨的无机纳米结构；荧光及磁性具有示踪及促进微球聚积功能；三维培养和bottom-up技术可以模拟体内微环境在体外构建组织工程复合体；3D打印技术可为不同缺损形态的CSD提供个性化修复重建方案。鉴于此，我们提出了通过构建具有仿ECM结构、荧光、磁性及骨诱导活性的多功能微球载体为支架材料，应用3D打印、三维培养及bottom-up技术，在体外预先制备出具有三维形态和个性化特征的组织工程骨复合体的系统性解决方案。并在裸鼠-大鼠-犬-灵长类动物实验中验证该方案的可行性，完成临床前研究。本项目将为实现组织工程技术在CSD个性化治疗的临床应用奠定基础。

由于缺少有效的策略同时修复骨和软骨组织，关节的骨软骨缺损修复目前仍是临床一大挑战。在本课题的资助下：（1）体外微球载体表征结果表明，壳聚糖/β-磷酸三钙（CS/β-TCP）纳米复合物微球具有良好的仿ECM结构, β-TCP纳米颗粒均匀分散在CS纤维网状结构中，较好的模仿了天然骨组织的结构与成分。 （2）体外细胞实验结果表明，CS/β-TCP微球（CTM）具有良好的生物相容性，骨髓间充质干细胞（BMSCs）能够在微球载体表面增殖且微球之间可通过BMSCs桥接在一起。（3）体外构建了CS/β-TCP@TGF-β1微球复合体（CTM-β1），细胞实验结果表明CTM能够促进BMSCs向成骨方向分化，CTM-β1能够促进BMSCs向成软骨方向分化。（4）体内构建SD大鼠膝关节骨软骨缺损模型，通过采用bottom-up策略通过将不同的微球组合植入到缺损部位，从而实现骨和软骨组织的同时修复。结果表明可以有效的修复大鼠膝关节的骨软骨缺损。通过本课题的研究，我们确定了CTM具有仿生的骨结构，可以作为干细胞的理想载体。此外由于CTM的ECM网状结构以及β-TCP对蛋白的高亲和性，CTM还可以作为生长因子载体，从而使得CTM具备多种功能。通过bottom-up策略，根据缺损部位的情况负载不同的细胞以及生长因子，从而实现多种组织的同时修复。