基于三周期极小曲面β-磷酸三钙仿生骨支架设计和生物活性的检测

Design and biological activity of beta-tricalcium phosphate biomimetic bone scaffold based on triply periodic minimal surfaces

背景:增材制造技术可以精确、个性化地定制具有复杂多孔结构的骨支架,达到恢复临界骨缺损区域松质骨结构和功能的效果。目的:通过材料学和细胞学表征,明确三周期极小曲面结构β-磷酸三钙生物陶瓷骨支架的机械性能及生物活性,揭示三周期极小曲面结构对成骨细胞的调控效果。方法:通过Matlab R2020a软件设计330,420,510μm 3种孔径的三周期极小曲面G曲面支架,Inspire 2018软件分析支架结构设计。以三周期极小曲面结构导出后的STL文件为蓝本,通过基于数字激光加工的增材制造技术制备3种β-磷酸三钙生物陶瓷支架,采用扫描电镜观测支架表面形貌,X射线衍射仪检测物相组成,万能材料试验机检测支架的力学强度。将MC3T3-E1细胞与3种支架共培养,检测细胞的增殖活性、黏附能力与碱性磷酸酶活性。结果与结论:(1)Inspire 2018软件显示,三周期极小曲面呈现出光滑、连续、均一的贯通式多孔结构;(2)扫描电镜下可见,基于数字激光加工的增材制造技术成功实现了三周期极小曲面结构的精确成型;(3)X射线衍射结果证实,支架由纯β-磷酸三钙晶相组成;(4)3组支架的压缩强度和弹性模量均处于或接近松质骨力学强度范围,且支架的压缩强度与孔径大小呈反比;(5)CCK8实验显示,MC3T3-E1细胞在3种支架上生长良好,生物活性与孔径呈现剂量依赖关系,510μm孔径促进细胞增殖效果最佳;(6)活细胞成像仪和激光共聚焦显微镜下可见,MC3T3-E1细胞能够在3种支架上实现早期黏附,且黏附量随孔径增大而增加;(7)碱性磷酸酶活性分析表明,420μm孔径支架上细胞的碱性磷酸酶活性最高;(8)结果表明,三周期极小曲面结构β-磷酸三钙生物陶瓷支架表现出较好的机械性能与生物活性,其中420μm孔径有利于促进细胞分化,510μm孔径有利于细胞增殖,具有修复临界骨缺损的应用潜力。

BACKGROUND:The bone scaffold with complex porous structure can be customized accurately and individually by additive manufacturing technology,so as to achieve the dual bionics of the structure and function of cancellous bone at critical-sized bone defects.OBJECTIVE:To clear the mechanical properties and biological activity ofβ-tricalcium phosphate bioceramic bone scaffold with triply periodic minimal surface structure,and reveal the regulatory effect of triply periodic minimal surface structure on osteoblasts through material and cytological characterizations.METHODS:Three kinds of triply periodic minimal surface G surface structureβ-tricalcium phosphate bioceramic bone scaffolds with different pore sizes of 330,420,and 510μm were designed by Matlab R2020 a software,and the design drawings were analyzed by Inspire 2018 software.The STL file exported based on the triply periodic minimal surface structure was the blueprint.β-Tricalcium phosphate scaffolds were fabricated by additive manufacturing technology based on digital laser processing.The surface morphology was observed by scanning electron microscope.The phase composition was detected by X-ray diffractometer,and the mechanical strength was detected by universal material testing machine.MC3 T3-E1 cells were co-cultured with scaffolds.Cell proliferation,cell adhesion,and alkaline phosphatase activity were detected.RESULTS AND CONCLUSION:(1)Inspire 2018 software showed that triply periodic minimal surface presented a smooth,continuous and uniform porous structure.(2)Scanning electron microscope confirmed that the additive manufacturing technology based on digital laser processing successfully realized the accurate molding of triply periodic minimal surface structure.(3)X-ray diffraction confirmed that the scaffold was composed of pureβ-tricalcium phosphate crystal phase.(4)The compressive strength and elastic modulus of the three groups of scaffolds were all within or near the range of cancellous bone,and the compressive strength of the scaffolds was inversely proportional to the pore size.(5)CCK8 assay showed that MC3 T3-E1 cells grew well on the scaffolds;the biological activity of the scaffolds was dose-dependent with the pore size;and the 510μm pore diameter scaffold had the best effect on promoting proliferation.(6)Living cell imager and laser confocal microscope showed that MC3 T3-E1 cells could adhere to the scaffold in the early stage,and the amount of adhesion increased with the increase of pore size.(7)Alkaline phosphatase activity analysis showed that the alkaline phosphatase activity of cells on 420μm scaffold was the highest.(8)The results show that the mechanical properties and biological activity of the triply periodic minimal surface structureβ-tricalcium phosphate bioceramic scaffold are excellent;the pore size of 420μm is beneficial to cell differentiation and 510μm is beneficial to cell proliferation,which has the potential to repair critical bone defects.