改良同轴3D打印具有空心管道结构的镁黄长石支架

Modified coaxial 3D-printed akermanite scaffold with hollow-pipe structure

背景:随着3D打印技术和骨组织工程的不断发展,越来越多的3D打印支架被运用到骨组织工程研究中,而具有中空管道结构的3D打印支架具有利于血管生长及营养物质输的优势,值得深入研究。目的:探讨镁黄长石释放的离子成分对大鼠骨髓间充质干细胞成骨分化及人脐静脉内皮细胞迁移的影响,并采用同轴3D打印技术制备具有中空管道结构的镁黄长石支架材料。方法:采用不同浓度梯度(1/4、1/8、1/16)的镁黄长石提取液分别培养大鼠骨髓干细胞和人脐静脉内皮细胞,通过CCK-8、碱性磷酸酶染色和RT-qPCR法检测大鼠骨髓间充质干细胞的增殖及成骨分化情况,通过Transwell实验观察不同浓度的镁黄长石提取液对内皮细胞迁移能力的影响。通过具有核/壳结构的改良3D打印系统制备具有中空管道结构的镁黄长石三维支架与实心镁黄长石三维支架,将支架浸泡于去离子水中,设定时间点提取浸提液,通过电感耦合等离子体原子发射光谱法检测离子释放浓度。结果与结论:(1)CCK-8实验显示,镁黄长石提取液呈浓度与时间依赖性促进骨髓间充质干细胞的增殖;(2)碱性磷酸酶染色显示,镁黄长石提取液呈浓度依赖性提高骨髓间充质干细胞的碱性磷酸酶活性;RT-qPCR检测显示,镁黄长石提取液呈浓度依赖性提高骨髓间充质干细胞碱性磷酸酶、Ⅰ型胶原及Rnux2 mRNA的表达;(3)镁黄长石提取液呈浓度依赖性促进内皮细胞的迁移;(4)将支架浸入去离子水中30 d,钙、镁、硅离子均呈现持续释放的特征,且具有中空管道结构镁黄长石三维支架的离子释放速率更快、浓度更高;(5)结果表明,采用改良同轴3D打印技术可制备具有空心管道结构的镁黄长石支架,其释放的生物活性离子成分对大鼠骨髓干细胞有明显的成骨诱导性,并可以趋化人脐静脉内皮细胞的迁移,促进血管再生。

BACKGROUND:With the continuous development of three-dimensional(3D)printing technology and bone tissue engineering,more and more 3D printed scaffolds have applied in study of bone tissue engineering.The 3D printed scaffold with hollow pipe structure has the advantages of vascular growth and nutrient delivery,which is worthy of further study.OBJECTIVE:To investigate the effect of ionic components released by akermanite on osteogenic differentiation of rat bone marrow mesenchymal stem cells and migration of human umbilical vein endothelial cells and fabricate the 3D-printed akermanite scaffold with hollow-pipe structure with coaxial 3D printing technology.METHODS:The rat bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were cultured with different concentrations of akermanite extracts(1/4,1/8 and 1/16).The proliferation of rat bone marrow mesenchymal stem cells was detected by CCK-8 assay.Osteogenic differentiation of human umbilical vein endothelial cells was investigated by alkaline phosphatase staining and RT-qPCR.Transwell assay was used to observe the migration of human umbilical vein endothelial cells in response to different concentrations of akermanite extracts.The akermanite scaffold with hollow-pipe structure was prepared using a modified printer nozzle with a core/shell structure with the printing system.The scaffolds were immersed into deionized water and the extracts were collected at set time points.The concentration of ionic products was detected with inductively coupled plasma atomic emission spectrometry.RESULTS AND CONCLUSIOIN:(1)CCK-8 assay indicated that the akermanite extracts had a concentration and time-related effect on promoting the proliferation of bone marrow mesenchymal stem cells.(2)Alkaline phosphatase staining showed that the akermanite extracts had a concentration-related effect on the alkaline phosphatase activity of rat bone marrow mesenchymal stem cells.RT-qPCR indicated that the extracts had a concentration-related effect on the mRNA expression of alkaline phosphatase,collagen-1 and Runx2 of bone marrow mesenchymal stem cells.(3)The akermanite extracts had a concentration-related effect on the migration of human umbilical vein endothelial cells.(4)The scaffolds would continuously release the Ca,Mg and Si ions during immersion into deionized water for 30 days.The akermanite scaffold with hollow pipes was conducive to the release of biologically active ions and had high concentration.(5)The result showed that the akermanite scaffolds with hollow pipes were successfully prepared with the modified 3D printer.The ionic products released by scaffolds had an obvious effect on osteogenesis of rat bone marrow mesenchymal stem cells and promoted migration of human umbilical vein endothelial cells,thereby accelerating vascular regeneration.