3D Printed Hierarchically Micro/nano Porous Bioactive Ceramics For Bone Regeneration

The hierarchical structure design of scaffolds plays an important role in bone regeneration.Micropores can provide the surface and space for the adhesion,proliferation and differentiation of related cells in bone regeneration,thus affect and regulate the mutual recognition and interaction between cells and materials significantly.Then these events can trigger specific biological reactions,activate the expression of related genes and ultimately affect the quality and efficiency of bone regeneration.However,the macro/micro pores of bioactive ceramic scaffolds can not be controlled accurately.Here,we designed and prepared hierarchical porous scaffolds containing macropores and nano micropores,and modulate the morphology of nanopores by using inorganic nanospheres and organic nanofibers respectively.The effect of hierarchical structure on promoting bone regeneration was studied from three aspects: osteogenesis,angiogenesis and immune regulation.We hope to provide a new therapy strategy for bone regeneration.In the first part,sacrificial method combined with three-dimensional printing technology was used to prepare macro & spherical microporous scaffold(MSMP scaffold)and macro & irregular microporous scaffold(MIMP scaffold).Firstly,the differences of pore morphology,porosity,specific surface area and surface roughness were studied.The results show that the scaffolds with nanopore structure have higher porosity,specific surface area and surface roughness.Compared the two different nanopore morphologies,MIMP scaffold was more significant.Based on these,the release of calcium and phosphorus ions,protein adsorption and cell adhesion of the hierarchical porous scaffolds were further studied.We found that MIMP scaffold could effectively accelerate the release of calcium and phosphorus ions,increase the amount of surface protein adsorption,and promote the cells adhesion onto its surface.In the second part,bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were cocultured with hierarchical porous scaffolds to verify the bone regenerative abilities.By detecting the expression levels of genes and proteins related to osteogenesis and angiogenesis,we studied the angiogenesis abilities in vitro,and further studied mechanism based on the behavior of macrophages modulate by scaffolds.The results showed that MIMP scaffold could effectively promote the differentiation of osteoblasts and enhance the osteogenic activity in vitro.In addition,MIMP scaffold can polarize macrophages to M2 phenotype to inhibit inflammation and create an immune microenvironment conducive to bone regeneration,which can positively regulate osteogenesis.In the third part,the New Zealand white rabbit tibia defect model was used for implantation experiment in vivo.Through qualitative and quantitative analysis of new bone formation,expression detection of angiogenesis and osteogenesis,macrophage related markers of cells extracted from living tissue,histological staining,which verified the results of the above in vitro study.All of the results further proved that the hierarchical porous scaffold has a positive effect of promoting bone regeneration.In conclusion,the hierarchical porous scaffolds containing nanopores were successfully prepared by the combination of three-dimensional printing technology and sacrificial templates method,which can control the morphology of nanopores on the scaffolds and provide an optimized structural microenvironment for bioactive ceramic scaffolds.The results of in vitro and in vivo show that the hierarchical porous structure of scaffolds accelerate the bone regeneration through ion dissolution and protein absorption.The results provide new ideas and strategies for the development of bone regeneration.