氧化石墨烯铜纳米颗粒修饰的中空管道支架诱导血管化骨再生的作用及其机制研究

Vascularization is critical for the biological function of large tissue-engineered bone. In previous studies we found scaffolds with multi-channels could accelerated the delivery of oxygen and nutrition to surviving cells, increased the vascularization process in the scaffolds. In addition, the coupling relationship between angiogenesis and osteogenesis were existed in the process of bone regeneration, while Hypoxia-inducible factor-1a(HIF-1α) played an important role in the process. Studies showed copper (Cu) could stimulate the expression of HIF-1α, and the up-regulation expression of osteogenic markers and the higher level of protein level. However, the high concentration of Cu induced cytotoxicity remains to be solved. By loading copper nanoparticles on Graphene oxide (GO-Cu) could achieve sustained and slow release of copper. It is not only started initial angiogenesis and osteogenesis, but also avoiding the cytotoxicity caused by long-term accumulation of copper. In this study, we prepared a hollow-struts-packed β-tricalcium phosphate (β-TCP) scaffolds with 3D printing strategy, then coating GO-Cu nanoparticles and loading bone marrow stromal cells (BMSCs) on the scaffold surface was made to stimulate BMSCs osteogenic differentiation and secret angiogenic inducing factor by GO-Cu to promoted rapid vascularization collaborate with the blood vessels ingrowthing along the pipeline. The loading BMSCs survival rate was improved feedbackly by the secret factor to better involving in bone regeneration. Finally, the effect of the vascularized bone regeneration was evaluated by in vivo study by animal models.

血管化是大块骨组织再生中亟需解决的难题。课题组研究发现具有管道设计的支架有利于营养扩散，促进组织细胞沿管道向材料深部长入，从而加速支架血管化。而在骨再生过程中，血管化与成骨之间存在耦合关系，低氧诱导因子1α（HIF-1α）在其中起了重要作用，研究发现铜离子（Cu）可以显著提高HIF-1α的表达，上调成血管、成骨相关基因和蛋白的表达，但高浓度Cu引起的细胞毒性还有待解决。通过氧化石墨烯负载铜纳米颗粒（GO-Cu）可以实现Cu的持续、缓慢释放，既启动初期局部成骨成血管活动，又避免Cu蓄积带来的细胞毒性。本课题通过3D打印技术制备β-TCP中空管状支架，表面进行GO-Cu涂层，并负载骨髓基质干细胞（BMSCs），通过GO-Cu刺激BMSCs成骨分化并分泌成血管诱导因子与沿管道长入的血管协同促进快速血管化，同时“反馈”提高BMSCs的生存率以更好的参与骨再生，最终在动物体内评估颌骨血管化再生效果。

随着生物材料和组织工程技术的发展，金属离子应用于生物材料的改性研究有了较大进展。铜（Cu）离子、锂（Li）离子作为人体内的微量元素，可以作为添加物，参与生物材料的螯合反应，增强机械强度，还能促进成骨细胞的增殖与分化。研究发现Cu离子可以显著提高HIF-1α的表达，上调成血管、成骨相关基因和蛋白的表达；Li离子也可以促进成骨细胞增殖、分化再生，对骨代谢进行调节。但高浓度离子引起的细胞毒性还有待解决。通过3D打印技术制备镁黄长石中空管状支架，可以实现离子的持续、缓慢释放，既启动初期局部成骨成血管活动，又避免离子蓄积带来的细胞毒性。本课题通过改良同轴三维打印机制备的镁黄长石支架具有空心管道结构，管道的外径为1mm，内径为500µm，抗压强度高达26Mpa，镁黄长石释放的离子成分促进了骨髓干细胞（BMSCs）的增殖、分化，干细胞的成骨及成血管相关基因表达升高，并趋化内皮细胞，促进血管生成。与实心的镁黄长石支架相比，空心管道结构更有利于营养物质的输送和生物活性离子的释放，负载的细胞具有更好的生物活性和成骨分化潜能。本实验过程中发现一定浓度的Li离子对BMSCs增殖及成骨分化具有促进作用，当Li离子浓度达到10mM时，BMSCs的细胞成活率显著下降；当Li离子浓度为5mM 时，其促成骨效果表现最佳，5mM锂离子能显著促进细胞内RUNX2、ALP、OCN和Col I等成骨相关转录因子的表达和OCN蛋白的表达。基于Li离子对BMSCs的影响，我们成功构建完成了Li离子修饰的中空管道的镁黄长石支架材料，探索了支架材料对离子的固载及控释影响，控制Li离子的缓释浓度。为Li离子修饰的中空管道3D支架材料修复体内兔颅骨缺损的效果评价提供体外数据支持，期待通过Li离子和中空管道支架二者的结合能够在大块颌骨缺损修复中取得理想的修复效果，为以后针对临床上肿瘤、创伤、感染造成的颌面部的大块骨缺损的治疗提供实验基础。