3D打印仿生磷灰石支架的成骨转化和再生研究

In the field of tissue engineering and regenerative medicine, it is always hot and critical for the construction of tissue and organ for transplantation in vitro. Herein, we propose to prepare three dimensional printed porous composite scaffolds using biomimetic apatite nanoparticles with element substitutions. After treating in continuous perfusion culture system, the printed biomimetic scaffolds are transformed into bone-like tissues under the continuous stream with bone marrow stem cells (BMSCs) and growth factors. The research involves: 1) Synthesis and characterization of biomimetic apatite nanoparticles using collagen and citric acid as templates and Zn/Sr as substitutions; 2) Fabrication of porous composite scaffolds using three dimensional printing; 3) Preparation of the scaffolds with BMP-2 and VEGF growth factors using polyelectrolyte multilayered coatings (chitosan/hyanuronic acid) to prolong the controlled release; 4) In the 3-dimensional continuous perfusion system, BMSCs are applied to the composite scaffolds continuously. The scaffolds are degraded and re-mineralization occurs to form new bone-like materials; 5) Bone integration of the obtained new implant bone-like materials are evaluated using the rabbit bone critical-size calvarial defect model. This research focuses on the combination of 3D printing and biomimetic self-assembly techniques, which is used for fabricating porous scaffolds with the biomimetic apatite particles. The resultant scaffolds are re-mineralized and transformed to bone-like tissue using BMSC under simulated circumstance. It will pave a new way to potentially create a novel large size engineered bone construct for implantation.

体外构建适用于移植的组织和器官一直是组织工程和再生医学领域的研究难点和热点。本项目拟将元素掺杂仿生磷灰石纳米粒子用三维打印制成多孔复合支架，并采用连续灌注培养系统使其转化为类骨组织材料。研究内容包括：1）用胶原和柠檬酸作为双模板，制备和表征掺杂Zn和Sr的磷灰石纳米粒子；2）用3D打印技术将仿生磷灰石纳米粒子制成多孔复合支架；3）用多层聚电解质（壳聚糖/透明质酸）修饰多孔支架，装载BMP-2和VEGF 生长因子，实现控释；4）在连续培养系统中，用BMSC细胞促使支架材料进行类骨转化，评价材料降解与再矿化程度；5）用兔的颅骨极限缺损模型评价转化的3D打印仿生材料的骨再生效果。本项目创新点在于：综合运用3D打印和仿生自组装，将仿生掺杂磷灰石支架通过细胞调控的再矿化过程转化成类骨组织，为新型大块骨植入材料的研究提供了新思路。

高活性人工骨植入支架材料一直是骨组织工程研究的热点。本项目主要研究了以元素掺杂仿生磷灰石纳米材料为打印墨水，使用3D生物打印技术制备多孔骨修复支架，通过兔颅骨极限尺寸模型评价了其骨组织再生修复的能力。结果如下：1)以胶原和柠檬酸作为调控矿化的模板组合，掺杂Zn或Sr元素、通过仿生矿化法制备了Zn或Sr掺杂的纳米磷灰石材料。选择Sr掺杂磷灰石材料制备3D打印浆料，发现其更易制成均匀的打印材料。3D打印多孔支架细胞相容性好，掺杂Sr元素后有利于细胞黏附。在兔颅骨极限尺寸（15mm直径）缺损修复模型中可以有效提升骨再生效果；2)改进3D生物打印浆料的配方，引入壳聚糖等聚电解质调节打印浆料的粘度，将打印的3D多孔支架中磷灰石无机物含量提高，抗压强度提升至与自体骨金标准相当；3）在3D打印支架上使用聚电解质层层自组装涂层改性，不仅提升了3D打印支架的力学性能，降低了支架的溶胀，还可作为载体缓释生长因子BMP-2和VEGF。动物实验结果表明载BMP-2和VEGF因子的3D打印复合支架显著可提高骨组织再生速度，并促进血管再生；4）使用GelMA作为打印原料替代明胶，通过同步光交联技术提升打印过程的稳定性，打印体出丝更加精细，打印体高度提升至55层，有利于后期打印大块骨植入材料的实施。项目发表SCI论文7篇，申请中国发明专利1项，相关研究成果为研制新型大块骨组织工程支架材料打下了坚实的基础。