生物打印具有微网络流体通道的三维结构
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摘要
背景:构建三维的预血管化系统,对于大尺寸、复杂三维结构组织内细胞的存活和功能表达均具有决定性作用。因此,寻找一种合适的预血管化策略已成为3D生物打印大尺寸、复杂三维组织亟待解决的问题。目的:对3D生物打印大尺寸、预血管化三维结构进行初步的探索。方法:用逆向工程软件Catia设计三维生物打印蓝图;以改装后的桌面级双喷头3D打印机为生物打印机,以聚乙烯醇为牺牲材料打印牺牲骨架;以大鼠骨髓间充质干细胞、海藻酸钠、琼脂糖和纳米纤维素溶液的混合物为细胞生物墨水。根据预先设计的参数进行生物打印,构建具有三维流体通道的组织工程三维结构体。观察打印获得的三维结构体,评价打印后及材料溶解后的细胞活性;体外培养打印获得的结构体,并用Alamar Blue试剂盒检测细胞在三维结构体中的增殖。结果与结论:利用Catia软件设计出了具有微孔的外壁及内部纵横交错的微管结构的双喷头打印模型,用该三维生物打印技术构建出具有自定义尺寸(尤其是高度)和预血管化的三维结构,结构体中细胞12h的存活率为(95.47±0.54)%,随着体外培养时间的延长,细胞存活率下降并稳定在80%以上。随着培养时间的增加,构建体中的细胞增殖呈上升趋势。结果表明:该生物打印方法可用于通过使用各种生物水凝胶材料制造不同特性的三维结构体,在生物制造具有临床相关尺寸的预血管化的复杂组织器官方面具有潜力。
BACKGROUND: Constructing a three-dimensional pre-vascularization system plays a decisive role in the survival and functional expression of cells in large and complex three-dimensional structures. Therefore, seeking a suitable pre-vascularization strategy has become a problem to be solved urgently in the 3 D bio-printing of large size and complex three-dimensional tissue.OBJECTIVE: To make a preliminary exploration on the 3 D bioprinting of large-scale and pre-vascularized three-dimensional structure. METHODS: Catia as a reverse engineering software was used to design a three-dimensional biological print blueprint. A modified desktop-grade dual-head 3 D printer was used as a biological printer and polyvinyl alcohol as a sacrificial material to print a sacrificial skeleton. The mixture of rat bone marrow mesenchymal stem cells, sodium alginate, agarose and nano-cellulose solution was used as cell biological ink. According to the pre-designed parameters for printing, the tissue engineering three-dimensional structure with three-dimensional fluid channel was constructed. The three-dimensional structure obtained by printing was observed to evaluate the cell activity after printing as well as after material dissolution. The printed structure was cultured in vitro and the proliferation of cells in the three-dimensional structure was detected by Alamar Blue kit. RESULTS AND CONCLUSION: A double-nozzle printing model with micro-porous outer wall and interlaced microtubules was designed by using Catia software. The three-dimensional biometric printing technology was used to construct the three-dimensional structure with self-defined size(especially its height) and pre-vascularization. The survival rate of the cells in the printed structure within 12 hours was(95.47±0.54)%, and as the in vitro culture time prolonged, the cell survival rate decreased but still exceeded 80%. Over time, the cell proliferation showed an increasing tendency. These findings indicate that this biometric printing method can be used to produce three-dimensional structures with different characteristics by using various bio-hydrogel materials, and has potential in the biofabrication of complex tissues and organs with clinically-related dimensions of pre-vascularization.
BACKGROUND: Constructing a three-dimensional pre-vascularization system plays a decisive role in the survival and functional expression of cells in large and complex three-dimensional structures. Therefore, seeking a suitable pre-vascularization strategy has become a problem to be solved urgently in the 3 D bio-printing of large size and complex three-dimensional tissue.OBJECTIVE: To make a preliminary exploration on the 3 D bioprinting of large-scale and pre-vascularized three-dimensional structure. METHODS: Catia as a reverse engineering software was used to design a three-dimensional biological print blueprint. A modified desktop-grade dual-head 3 D printer was used as a biological printer and polyvinyl alcohol as a sacrificial material to print a sacrificial skeleton. The mixture of rat bone marrow mesenchymal stem cells, sodium alginate, agarose and nano-cellulose solution was used as cell biological ink. According to the pre-designed parameters for printing, the tissue engineering three-dimensional structure with three-dimensional fluid channel was constructed. The three-dimensional structure obtained by printing was observed to evaluate the cell activity after printing as well as after material dissolution. The printed structure was cultured in vitro and the proliferation of cells in the three-dimensional structure was detected by Alamar Blue kit. RESULTS AND CONCLUSION: A double-nozzle printing model with micro-porous outer wall and interlaced microtubules was designed by using Catia software. The three-dimensional biometric printing technology was used to construct the three-dimensional structure with self-defined size(especially its height) and pre-vascularization. The survival rate of the cells in the printed structure within 12 hours was(95.47±0.54)%, and as the in vitro culture time prolonged, the cell survival rate decreased but still exceeded 80%. Over time, the cell proliferation showed an increasing tendency. These findings indicate that this biometric printing method can be used to produce three-dimensional structures with different characteristics by using various bio-hydrogel materials, and has potential in the biofabrication of complex tissues and organs with clinically-related dimensions of pre-vascularization.
引文
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[27]Kundu J,Shim JH,Jang J,et al.An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.J Tissue Eng Regen Med.2015;9(11):1286-1297.
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[33]Richards D,Jia J,Yost M,et al.3D Bioprinting for Vascularized Tissue Fabrication.Ann Biomed Eng.2017;45(1):132-147.
[34]Fedorovich NE,Swennen I,Girones J,et al.Evaluation of photo crosslinked lutrol hydrogel for tissue printing applications.Biomacromolecules.2009;10(7):1689-1696.
[35]宋杨,王晓飞,王宇光,等.人脂肪间充质干细胞与生物材料共混物三维打印体的体内成骨[J].北京大学学报(医学版),2016,48(1):45-50.
[36]薛世华,吕培军,王勇,等.人牙髓细胞共混物三维生物打印技术[J].北京大学学报(医学版),2013,45(1):105-108.
[37]K?pf M,Campos DF,Blaeser A,et al.A tailored three-dimensionally printable agarose-collagen blend allows encapsulation,spreading,and attachment of human umbilical artery smooth muscle cells.Biofabrication.2016;8(2):025011.
[38]Wu Z,Xin S,Xu Y,et al.Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation.Sci Rep.2016;6:24474.
[2]Peltola SM,Melchels FP,Grijpma DW,et al.A review of rapid prototyping techniques for tissue engineering purposes.Ann Med.2008;40(4):268-280.
[3]Mironov V,Trusk T,Kasyanov V,et al.Biofabrication:a 21st century manufacturing paradigm.Biofabrication.2009;1(2):1-16.
[4]Konig G,McAllister TN,Dusserre N,et al.Mechanical propertie of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.Biomaterials.2009;30(8:1542-1550.
[5]Wang X,Yan Y,Zhang R.Recent trends and challenges in complex organ manufacturing.Tissue Eng Part B Rev.2010;16:2:189-197.
[6]Wüst S,Müller R,Hofmann S.3D Bioprinting of complex channels-Effects of material,orientation,geometry,and cell embedding.J Biomed Mater Res A.2015;103(8):2558-2570.
[7]Pati F,Ha DH,Jang J,et al.Biomimetic 3D tissue printing for soft tissue regeneration.Biomaterials.2015;62:164-175.
[8]Das S,Pati F,Choi YJ,et al.Bioprintable,cell-laden silk fibroin-gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs.Acta Biomater.2015;11:233-246.
[9]Gao Q,He Y,Fu JZ,et al.Coaxial nozzle-assisted 3Dbioprinting with built-in microchannels for nutrients delivery.Biomaterials.2015;61:203-215.
[10]Duarte CDF,Blaeser A,Buellesbach K,et al.Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering.Adv Healthc Mater.2016;5:11:1336-45.
[11]Tan YJ,Tan X,Yeong WY,et al.Hybrid microscaffold-based 3Dbioprinting of multi-cellular constructs with high compressive strength:A new biofabrication strategy.Sci Rep.2016;6:39140
[12]Colosi C,Shin SR,Manoharan V,et al.Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink.Adv Mater.2016;28:4:677-684.
[13]Blanchette CD,Knipe JM,Stolaroff JK,et al.Printable enzyme-embedded materials for methane to methanol conversion.Nat Commun.2016;7:11900.
[14]He Y,Yang F,Zhao H,et al.Research on the printability of hydrogels in 3D bioprinting.Sci Rep.2016;6:29977.
[15]Yu Y,Moncal KK,Li J,et al.Three-dimensional bioprinting usin self-assembling scalable scaffold-free“tissue strands”as a new bioink.Sci Rep.2016;6:28714.
[16]臧晓龙,孙健,李亚莉,等.3D生物打印构建聚乳酸羟基乙酸/纳米羟基磷灰石支架骨形态发生蛋白2缓释复合体的实验研究[J].中国组织工程研究,2016,20(16):2405-2411.
[17]宋杨,王晓飞,王宇光,等.人脂肪间充质干细胞与生物材料共混物三维打印体的体内成骨[J].北京大学学报(医学版),2016,48(1):45-50.
[18]Jang J,Park HJ,Kim SW,et al.3D printed complex tissue construct using stem cell-laden decellularized extracellular matrix bioinks for cardiac repair.Biomaterials.2017;112:264-274.
[19]Carvalho AF,Gasperini L,Ribeiro RS,et al.Control of osmotic pressure to improve cell viability in cell-laden tissue engineering constructs.J Tissue Eng Regen Med.2018;12(2):e1063-e1067.
[20]Costantini M,Testa S,Mozetic P,et al.Microfluidic-enhanced3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo.Biomaterials.2017;131:98-110.
[21]Ong CS,Fukunishi T,Zhang H,et al.Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.Sci Rep.2017;7(1):4566.
[22]Chrobak KM,Potter DR,Tien J.Formation of perfused,functional microvascular tubes in vitro.Microvasc Res.2006;71(3):185-196.
[23]Ozbolat IT,Yu Y.Bioprinting toward organ fabrication:challenges and future trends.IEEE Trans Biomed Eng.2013;60(3):691-699.
[24]Paulsen SJ,Miller JS.Tissue vascularization through 3Dprinting:Will technology bring us flow?Dev Dyn.2015;244(5):629-640.
[25]Lee VK,Kim DY,Ngo H,et al.Creating perfused functional vascular channels using 3D bio-printing technology.Biomaterials.2014;35(28):8092-8102.
[26]杨道朋,夏旭.3D打印生物材料研究及其临床应用优势[J].中国组织工程研究,2017,21(18):2927-2933.
[27]Kundu J,Shim JH,Jang J,et al.An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.J Tissue Eng Regen Med.2015;9(11):1286-1297.
[28]刘伟,刘萌,祝劲松,等.人骨髓间充质干细胞的体外培养、鉴定及成骨分化[J].中国组织工程究,2012,16(14):2515-2519.
[29]Ling Y,Rubin J,Deng Y,et al.A cell-laden microfluidic hydrogel.Lab Chip.2007;7(6):756-762.
[30]Novosel EC,Kleinhans C,Kluger PJ.Vascularization is the key challenge in tissue engineering.Adv Drug Deliv Rev.2011;63:(4-5):300-311.
[31]Bertassoni LE,Cecconi M,Manoharan V,et al.Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs.Lab Chip.2014;14(13):2202-2211.
[32]Kolesky DB,Homan KA,Skylar-Scott MA,et al.Three-dimensional bioprinting of thick vascularized tissues.Proc Natl Acad Sci U S A.2016;113(12):3179-3184.
[33]Richards D,Jia J,Yost M,et al.3D Bioprinting for Vascularized Tissue Fabrication.Ann Biomed Eng.2017;45(1):132-147.
[34]Fedorovich NE,Swennen I,Girones J,et al.Evaluation of photo crosslinked lutrol hydrogel for tissue printing applications.Biomacromolecules.2009;10(7):1689-1696.
[35]宋杨,王晓飞,王宇光,等.人脂肪间充质干细胞与生物材料共混物三维打印体的体内成骨[J].北京大学学报(医学版),2016,48(1):45-50.
[36]薛世华,吕培军,王勇,等.人牙髓细胞共混物三维生物打印技术[J].北京大学学报(医学版),2013,45(1):105-108.
[37]K?pf M,Campos DF,Blaeser A,et al.A tailored three-dimensionally printable agarose-collagen blend allows encapsulation,spreading,and attachment of human umbilical artery smooth muscle cells.Biofabrication.2016;8(2):025011.
[38]Wu Z,Xin S,Xu Y,et al.Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation.Sci Rep.2016;6:24474.