3D生物打印对明胶/海藻酸钠/硅酸镁锂复合细胞水凝胶支架成骨分化的影响  

Effect of 3D bioprinting on osteogenic differentiation of gelatin/sodiuni alginate/laponite composite cellular hydrogel scaffolds

作　　者：苗胜 周津如 雷星[3] 刘斌 程朋真 裴国献 毕龙 Miao Sheng;Zhou Jinru;Lei Xing;Liu Bin;Cheng Pengzhen;Pei Gaoxian;Bi Long(Department of Orthopedics,Xijing Hospital,Air Force Military,Medical University,Xi’an 710032,China;Department of Radiology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471000,Chirui;Department of Orthopedics,Linyi People's Hospital,Linyi 276002,China;Department of Orthopedics,General Hospital of Northern Theater Command of PLA,Shenyang 110015,China;Research Institute of Orthopedics of PLA,Air Force Military Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学西京医院骨科,西安710032 [2]郑州大学附属洛阳中心医院影像科,471000 [3]山东省临沂市人民医院骨科,276002 [4]中国人民解放军北部战区总医院骨科,沈阳110015 [5]空军军医大学全军骨科研究所,西安710032

出　　处：《中华创伤杂志》2021年第10期938-946,共9页Chinese Journal of Trauma

基　　金：国家重点研发专项(2016YFC1100304);国家自然科学基金(81672189,81902202)。

摘　　要：目的应用3D生物打印技术制备明胶/海藻酸钠/硅酸镁锂复合水凝胶负载骨髓间充质干细胞(BMSCs)的仿生活性组织工程支架,探讨3D生物打印对含BMSCs水凝胶支架的成骨分化作用。方法常规提取2周龄SD大鼠BMSCs并用流式细胞术鉴定,将明胶、海藻酸钠、硅酸镁锂混合,加入BMSCs后应用3D生物打印技术制备含细胞的复合水凝胶支架,应用注模法制备含细胞的非打印支架。体外实验中根据是否打印分为含细胞打印组和含细胞非打印组,每组制备12个样品,并设置单纯细胞对照组。通过扫描电镜观察复合水凝胶内部结构,冻干法测定支架膨胀率和含水量。在培养3 d后用鬼笔环肽染色观察支架内细胞活性,分别于培养1,3,7 d后进行细胞增殖与毒性检测(CCK-8)实验测定吸光度值确定细胞增殖情况和培养7,14 d后使用RT-PCR检测成骨细胞特异性转录因子(Osterix)、骨钙素(OCN)、Ⅰ型胶原等成骨相关基因表达。体内实验根据是否打印及是否含有细胞分四组:含细胞打印植入组、含细胞非打印植入组、不含细胞打印植入组、不含细胞非打印植入组,每组支架制备9个样品。将四组支架分别植入36只8周龄SD大鼠臀后侧肌袋,左右随机,术后2,4,8周取材分别拍摄X线片,并对8周取材组织进行HE及Masson染色观察成骨分化情况。结果流式细胞术鉴定细胞为BMSCs。水凝胶内部孔隙明显,细胞在孔隙内可自由伸展。含细胞打印组的支架膨胀率为(1 039.37±30.66)%,支架含水量为(91.21±0.26)%,与含细胞非打印组的支架膨胀率[(1 032.38±35.05)%]和支架含水量[(91.16±0.28)%]比较,差异无统计学意义(P>0.05)。在体外培养3 d水凝胶内细胞生长状态良好。体外培养1 d,各组吸光度值差异无统计学意义(P>0.05);培养3 d时,含细胞打印组和含细胞非打印组吸光度值差异无统计学意义(P>0.05),但均高于单纯细胞对照组(P<0.05);培养7 d后,含细胞打Objective To prepare hiomimetic tissue engineering scaffolds of gelatin/sodium alginale/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique,and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing hone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were routinely extracted and identified hy flow cytometry.Gelatin,sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting.Non-printed scaffolds containing cells were prepared by injection molding method.In vitro,the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed,with 12 samples per group.Another simple cell culture group was set as control.Then,the internal structure of the composite hydrogel was observed hy scanning electron microscope,and the expansion rate and water content of the scaffolds were measured by freeze-drying method.At day 3 after culture,the growth status of RMSCs was observed by phalloidine staining,cell counting kit(CCK)-8 assay was used to detect cell activity in scaffolds at days 1,3,and 7 after culture and KT-PCR to detect the expression of osteogenesis related genes Osterix,osteocalcin(OCN)and collagen 1 at days 7 and 14 ofter culture.In vivo,four groups were set according to printing or not and whether containing cells or not:printing important group with cells,non-printing implant group with cells,printing implant group without cells and non-printing implant group without cells,with 9 samples per group.Seaffolds in four groups were implanted to the posterior gluteal muscle pouches(random on left or right)of 368-week-old SD rats,respectively.The samples were taken X-ray images at 2,4 and 8 weeks after operation,respectively.The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining.Results The flow cytometry,showed that the cells were BMSCs.Internal pores of hydrogels were obviou

关 键 词：打印 三维 水凝胶 间充质干细胞 成骨分化 

分 类 号：R318.08[医药卫生—生物医学工程]