双喷头协同三维打印人脂肪间充质干细胞复合成骨支架的研究  

A preliminary study on osteogenic differentiation of double nozzle co-bioprinting human adipose-derived mesenchymal stem cells 3D scaffold

作　　者：李博 李箐[1] 郭亚娟 潘硕 吕培军[1] 王勇[1] LI Bo;LI Qing;GUO Ya-juan;PAN Shuo;LYU Pei-jun;WANG Yong(Center of Digital Dentistry,Peking University School And Hospital Of Stomatology&National Center of Stomatology&National Engineer Laboratory For Digital And Material Technology,Beijing 100081,China)

机构地区：[1]北京大学口腔医院,口腔医学数字化研究中心,国家口腔医学中心,国家口腔疾病临床医学研究中心,口腔数字化医疗技术和材料国家工程实验室,北京100081 [2]国家药品监督管理局医疗器械技术审批中心,北京100081

出　　处：《中华老年口腔医学杂志》2022年第1期11-16,共6页Chinese Journal of Geriatric Dentistry

基　　金：北京大学口腔医院基金(项目编号:PKUSS20200113)。

摘　　要：目的:初步研究聚己内酯(Polycaprolactone,PCL)与包裹有人脂肪间充质干细胞(Human Adipose-Derived Mesenchymal Stem Cells,hASCs)的水凝胶双喷头协同三维生物打印支架的力学性能,以及在纳米羟基磷灰石(Nano Hydroxyapatite,nHA)的参与下对细胞增殖和成骨分化的影响。方法:利用计算机设计支架模型,以细胞-海藻酸钠-明胶/PCL(AGP组)进行双喷头三维生物打印,细胞-海藻酸钠-明胶(AG组)单喷头打印,并在两组支架中分别添加nHA,探索双喷头打印的参数,在体外测试两组支架的力学性能。对两组支架进行CCK-8检测、CAM/PI染色和RT-PCR检测,分析双喷头打印支架中细胞的增殖和成骨分化能力。结果:调试打印机压力、速度、温度等参数后,可打印结构稳定的三维支架;力学性能实验中,AGP组弹性模量大于AG组(P<0.05);CCK-8和CAM/PI染色结果显示AG组和AGP组打印体内hASCs在7d时具有良好的增殖能力和细胞存活率。PCR结果显示,打印完成后14天,水凝胶中添加了纳米羟基磷灰石的AG组和AGP组成骨相关基因RUNX2、骨钙素(osteocalcin,OCN)和osterix(OSX)表达显著高于不添加纳米羟基磷灰石的AG组。结论:在适宜的打印参数下可打印出力学强度良好的双喷头三维支架,熔融PCL与水凝胶共同打印不会影响hASCs的增殖与存活率,纳米羟基磷灰石加入水凝胶中可提高hASCs的体外成骨分化能力。Objective:To study the mechanical properties of double nozzle co-bioprinting3D scaffold consisting of polyca-prolactone and hydrogel with human adipose-derived mesenchymal stem cells(hASCs),and the effects of nano-hydroxyapatite(nHA)on cell proliferation and osteogenic differentiation.Methods:The scaffold model was designed by computer,and the double-nozzle bio-printing of cell-sodium alginate-gelatin/PCL(AGP group)was performed as the experimental group,cell-sodium alginate-gelatin(AG group)was directly printed with a single nozzle as the control group to explore the parameters of double-nozzle printing,and to test the mechanical properties of the two groups of scaffolds in vitro.CCK-8,CAM/PI staining and RT-PCR were performed on the two scaffolds to analyze the proliferation and osteogenic differentiation of the cells in the scaffolds.Results:After adjusting the pressure,speed,temperature,the 3d composite scaffolds with stable structure can be printed.In the mechanical properties experiment,the elastic modulus of AGP group was greater than AG group(P<0.05).CCK-8 and CAM/PI staining results showed that AGP group and AG group had a good proliferation and cell viability of hASCs at 7 days after being printed.PCR results showed that 14 days after the printing,the expression of osteoporosis-related genes(RUNX2,osteocalcin and osterix)were significantly higher inthe AG group and AGP groupwith nano hydroxyapatite added to the hydrogel than the AG group without nano hydroxyapatite added.Conclusion:The double nozzle composite scaffolds with good mechanical strength can be printed with appropriate printing parameters and proportions.The co-printed of molten PCL and hydrogel will not affect the proliferation and viability of hASCs.The addition of nano-hydroxyapatite into hydrogel can increase osteogenic differentiation of hASCs in vitro.

关 键 词：三维生物打印 水凝胶 聚己内酯 纳米羟基磷灰石 

分 类 号：R782[医药卫生—口腔医学]