3D打印肺混合细胞结构体植入兔体内研究
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摘要
目的通过将三维(3D)生物打印的肺混合细胞水凝胶结构体片段植入兔背部皮下,探讨类肺组织结构体片段植入的可行性。方法采用胰蛋白酶消化法提取新生兔原代肺混合细胞。3D生物打印肺混合细胞-海藻酸钠-明胶3D水凝胶网格状结构体片段,活/死细胞双荧光染色观察细胞存活率。并将该结构体植入兔背部皮下。植入2周后,取出结构体片段,并进行组织病理学检测类肺组织结构形成情况。结果打印后的肺混合细胞在三维结构体中的存活率为83%±2%。兔肺细胞水凝胶支架体内植入2周后,可见植入物尚未降解。通过HE染色与Masson染色观察,表明植入物中肺细胞均匀分布,未见细胞变性及死亡。结论 3D生物打印的肺结构体具有再造类肺组织片段的潜力,将于基础医学和临床之间搭建桥梁,为肺脏再生打下基础。
Objective Three dimensional( 3 D) bioprinted lung-like hydrogel structure lamellas were implanted in the rabbits' backs to discuss its feasibility of implanting lung-like structure lamellas. Methods Lung primary blend cells were extracted from lung tissue pieces of newborn rabbit by 0. 25% trypsin digestion. 3 D structure lamella containing rabbit lung blend cells-alginate-gelatin blend was constructed using 3 D bioprinting technique. Then the cells' survival rate was detected by live-dead cell double fluorescence staining. The 3 D structure lamellas were implanted in the rabbits' backs. At 14 days after implantation,histopathological changes of the structure lamellas were examined by hematoxylin-eosin( HE) stain and Masson trichrome stain. Results The live/dead cell fluorescence staining results showed that the lung blend cells' survival rate was about 83% ± 2% after printing. The hydrogel scaffolds were not degraded at 14 days after implantation. Results of histological observation showed that the lung cells were evenly distributed in the hydrogel scaffolds,and that cell degeneration and death did not happen. Conclusion Bioprinted lung structure lamella has the potential to regenerate lung-like tissue structure. Our results demonstrated that it's possible to build a bridge between basic medicine and clinical practice,and lay the foundation for regeneration of the lung structure lamella.
Objective Three dimensional( 3 D) bioprinted lung-like hydrogel structure lamellas were implanted in the rabbits' backs to discuss its feasibility of implanting lung-like structure lamellas. Methods Lung primary blend cells were extracted from lung tissue pieces of newborn rabbit by 0. 25% trypsin digestion. 3 D structure lamella containing rabbit lung blend cells-alginate-gelatin blend was constructed using 3 D bioprinting technique. Then the cells' survival rate was detected by live-dead cell double fluorescence staining. The 3 D structure lamellas were implanted in the rabbits' backs. At 14 days after implantation,histopathological changes of the structure lamellas were examined by hematoxylin-eosin( HE) stain and Masson trichrome stain. Results The live/dead cell fluorescence staining results showed that the lung blend cells' survival rate was about 83% ± 2% after printing. The hydrogel scaffolds were not degraded at 14 days after implantation. Results of histological observation showed that the lung cells were evenly distributed in the hydrogel scaffolds,and that cell degeneration and death did not happen. Conclusion Bioprinted lung structure lamella has the potential to regenerate lung-like tissue structure. Our results demonstrated that it's possible to build a bridge between basic medicine and clinical practice,and lay the foundation for regeneration of the lung structure lamella.
引文
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18 Tan Q,Choi KM,Sicard D,et al. Human airway organoid engineering as a step toward lung regeneration and disease modeling[J]. Biomaterials,2017,113(1):118-132
19 Dye BR,Hill DR,Ferguson MA,et al. In vitro generation of human pluripotent stem cell derived lung organoids[J]. Elife,2015,4:e05098
2 Nichols JE,Niles JA,Vega SP,et al. Modeling the lung:Design and development of tissue engineered macro-and micro-physiologic lung models for research use[J]. Exp Biol Med(Maywood),2014,239(9):1135-1169
3 Kenzaki K,Sakiyama S,Kondo K,et al. Lung regeneration:implantation of fetal rat lung fragments into adult rat lung parenchyma[J]. J Thorac Cardiovasc Surg,2006,131(5):1148-1153
4 Chun YW,Crowder SW,Mehl SC,et al. Therapeutic application of nanotechnology in cardiovascular and pulmonary regeneration[J].Comput Struct Biotechnol,2013,7(8):e201304005
5 Riquet M,Mordant P,Pricopi C,et al. A review of 250 ten-year survivors after pneumonectomy for non-small-cell lung cancer[J].Eur J Cardiothorac Surg,2014,45(5):876-881
6 Petersen TH,Calle EA,Zhao L,et al. Tissue-engineered lungs for in vivo implantation[J]. Science,2010,329(5991):538-541
7 Lau AN,Goodwin M,Kim CF,et al. Stem cells and regenerative medicine in lung biology and diseases[J]. Mol Ther,2012,20(6):1116-1130
8 罗涛,唐靓,杨亚冬,等. 3D生物打印技术在构建兔原代肝细胞水凝胶结构体片段上的研究[J].中国卫生检验杂志,2017,27(18):2596-2599
9 Li X,Cai H,Cui X,et al. Prevention of late postpneumonectomy complications using a 3D printed lung in dog models[J]. Eur J CardioThorac Surg,2014,46(5):e67-e73
10 You F,Wu X,Chen X. 3D printing of porous alginate/gelatin hydrogel scaffolds and their mechanical property characterization[J]. Int J Polymeric Mater Polymeric Biomater,2017,66(6):299-306
11 Nichols JE,Niles JA,Cortiella J. Design and development of tissue engineered lung:progress and challenges[J]. Organogenesis,2009,5(2):57-61
12 Mandrycky C,Wang Z,Kim K,et al. 3D bioprinting for engineering complex tissues[J]. Biotechnol Adv,2016,34(4):422-434
13 Wilkinson DC,Alva-Ornelas JA,Sucre JM,et al. Development of a three-dimensional bioengineering technology to generate lung tissue for personalized disease modeling[J]. Stem Cells Transl Med,2017,6(2):622-633
14 Horváth L,Umehara Y,Jud C,et al. Engineering an in vitro airblood barrier by 3D bioprinting[J]. Sci Rep,2015,5:7974
15 Shah SB,Singh A. Cellular self-assembly and biomaterials-based organoid models of development and diseases[J]. Acta Biomater,2017,53(1):29-45
16 Radenkovic D,Solouk A,Seifalian A. Personalized development of human organs using 3D printing technology[J]. Med Hypotheses,2016,87(1):30-33
17 Zhao Y,Li Y,Mao S,et al. The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology[J]. Biofabrication,2015,7(4):045002
18 Tan Q,Choi KM,Sicard D,et al. Human airway organoid engineering as a step toward lung regeneration and disease modeling[J]. Biomaterials,2017,113(1):118-132
19 Dye BR,Hill DR,Ferguson MA,et al. In vitro generation of human pluripotent stem cell derived lung organoids[J]. Elife,2015,4:e05098