Coaxial printed double crosslinked alginate/silk fibroin vascular network scaffold

doi:10.3969/j.issn.2095-4344.1733

Abstract

Abstract:

BACKGROUND: Construction of tissue engineering vascular structures is a key to the regeneration of complex tissues and organs. The use of three-dimensional printing technology to construct vascular structures has become a hotspot.

OBJECTIVE: A highly connected and perfusable vascular network was rapidly deposited by a three-dimensional bioprinter with a coaxial nozzle using alginate/silk fibroin bioink.

METHODS: A mixed solution containing 5% sodium alginate and 5% silk fibroin as the bio-ink, and a mixed solution containing 5% calcium chloride and 13% F127 as the crosslinker, alginate/silk fibroin gel was printed using a bio-printer and subjected to optical coherence tomography and scanning electron microscopy. The solution containing 5% sodium alginate and 5% silk fibroin mixed with the suspension of human liver cancer cells (C3A) as the bio-ink, and a mixed solution containing 5% calcium chloride and 13% F127 as the crosslinker, the cell-containing sodium alginate/silk fibroin gel was printed on a bio-printer. Then, the printed scaffold was placed in the medium for 24 hours, and then stained by Calcein-AM and observed under a fluorescence microscope.

RESULTS AND CONCLUSION: (1) Optical coherence tomography: a multi-layer composite hollow tube network formed the structure of the scaffold. The gel filament had a complete hollow structure, and the channels were interconnected. The structure was similar to the hollow passage of the blood vessel, which was beneficial to the transportation of nutrients and metabolic waste. (2) Scanning electron microscope: the hollow pipe structure with the diameter of about 400 μm arranged in parallel, and the pipe boundaries were clear. The microporous structure caused by the removal of F127 was formed between the boundaries. (3) Fluorescence microscope: the cells were evenly dispersed in the material on both sides of the channel, and the cells grew well in the scaffold with the survival rate of above 95%. (4) These results suggest that three-dimensional bioprinting technology based on coaxial nozzles and sodium alginate/silk fibroin bio-ink can be used to construct vascularized functional tissues in the future.

Key words: alginate, silk fibroin, three-dimensional bioprinting, coaxial nozzle, hollow channel structure, bio-ink, double crosslinked network, perfusable vascular network

CLC Number:

Li Ningning, Xu Mingen, Suo Hairui, Wang Ling. Coaxial printed double crosslinked alginate/silk fibroin vascular network scaffold[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(18): 2865-2871.

Figures/Tables 5

2.1 材料流变性能 3D细胞打印是将细胞与生物材料混合成的生物墨水打印成预先设计的3D结构，因此材料自身的流变性能及混合细胞后的流变性能对生物墨水的可打印性至关重要。图2A为材料的黏度曲线，发现对比单纯海藻酸钠材料，海藻酸钠/丝素蛋白复合材料具有明显的剪切变烯特性，更有利于从喷头中挤出，从而在挤出过程中减少对细胞的损伤；而加入细胞后2种材料的黏度并无明显变化，说明细胞的加入对打印参数影响不大。海藻酸钠/丝素蛋白复合材料的黏度范围为3.07-22 Pa•s，与脱细胞外基质的黏度(2.8-23.6 Pa•s)相似[27]，有利于保持细胞活性。血管壁属于生物软组织，其最大的特点就是具有黏弹性。图2B，C中，有无细胞材料的黏弹性测量曲线显示出相似的趋势，将细胞与材料混合后，其储能模量(G')和损耗模量(G'')有轻微降低，这是因为与水凝胶相比，细胞可被认为是软颗粒，因此嵌入细胞会使水凝胶的网络结构发生轻微的变化[28]。在角速度为100 r/s的流变测试中，交联后海藻酸钠水凝胶支架的储存模量为19.7 kPa，损耗模量为 3.36 kPa；而交联后海藻酸钠/丝素蛋白水凝胶支架的储能模量为199.0 kPa，损耗模量为29.9 kPa，较交联后海藻酸钠水凝胶支架的测试结果有显著提高(P < 0.05)。复合剪切模量由剪切储存模量(G')和剪切损耗模量(G'')决定，复合剪切模量的绝对值能够表示在动态条件下水凝胶的硬度。支架需要具有的机械性能，除适宜细胞生长外，也要保证三维结构在处理、体外培养过程中维持结构的稳定性[29]。相比单纯海藻酸钠水凝胶支架，复合水凝胶支架的储存和损耗模量都有所增加则，其复合剪切模量绝对值也相应增加，表明复合水凝胶的硬度大于纯海藻酸钠水凝胶；相对于纯海藻酸钠支架，复合水凝胶支架更足以承受内部和外部机械载荷的强度，来确保生物打印结构体的稳定性，这源于复合凝胶中钙离子螯合交联海藻酸钠和丝素蛋白形成的β折叠结构，形成的双交联网络。"

2.2 支架的打印及工艺参数优化利用同轴喷头进行3D打印的工艺参数，包括材料的挤出速率、喷头的移动速度等。打印前，将同轴喷头固定在生物3D打印机的移动轴上，设置合适的注射泵流速和打印速度，生物墨水在针尖处和交联剂接触反应(图1b)，形成具有一定机械强度的凝胶中空丝[30]，打印机移动轴带动同轴喷头按照预先规划好的打印路径运动，实现3D结构的叠层制造。同轴喷头外管中的水凝胶溶液被交联，形成管壁，交联剂通过内管在生物墨水中流通形成中空通道。交联剂的离子完全渗透进水凝胶需要一定的时间，取决于交联剂离子浓度、海藻酸钠溶液浓度、管壁厚度等。因此，刚挤出的中空纤维内层先被交联，而外层未交联，利用未交联的外层实现丝之间的黏合，以保证支架结构的稳定性。生物墨水与交联剂挤出速率不匹配时会导致材料堆积，使打印的管道出现内外壁不光滑的现象，如图3A所示；在出丝速度过快而喷头移动速度过慢时，会出现管道弯曲的现象，如图3B所示；当前2种情况同时存在时，打印结果如图3C所示，通道弯曲且内外壁都有材料堆积。经调试，设定生物墨水的挤出速率为7 μL/s、交联剂溶液挤出速率为5 μL/s时，通过测试单位时间丝的垂直挤出长度，得到出丝速度为11 mm/s，于是设定打印速度与出丝速度同为11 mm/s，与之匹配的打印层高设为0.65 mm，间距设为 2 mm。合适的层高可避免破坏已打印的结构，并能够维持通道结构的形状和完整性；而合适的打印间距能够保证支架具有高的孔隙率，利于氧气和营养物质的运输及代谢废物的排出。参数调试后打印出的2层通道如图3D所示，管壁光滑且孔隙均匀。通过图3E的4层通道图可看出，采用间隔层错位打印的方法，使同向的路径相互交错，达到了提高孔隙精度的目的。"

References

[1]Battistella E,Varoni E,Cochis A,et al.Degradable polymers may improve dental practice J Appl Biomater Biomech. 2011;9(3):223-231.

[2]Lee W,Debasitis JC, Lee VK,et al.Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.Biomaterials. 2009;30(8):1587-1595.

[3]Yang EK,Seo YK,Youn HH,et al.Tissue engineered artificial skin composed of dermis and epidermis. Artif Organs. 2000;24(1):7-17.

[4]陈红.英国研究出3D打印人类眼角膜新材料[J].计算机与网络, 2018,44(12): 10.

[5]Lee W,Lee V,Polio S,et al.On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels. Biotechnol Bioeng.2010;105(6):1178-1186.

[6]Hribar KC,Soman P,Warner J,et al.Light-assisted direct-write of 3D functional biomaterials. Lab Chip.2013;14(2):268-275.

[7]Yuichi N,Makoto N,Chizuka H,et al.Development of a three-dimensional bioprinter: construction of cell supporting structures using hydrogel and state-of-the-art inkjet technology.J Biomech Eng.2009;131(3):35001.

[8]Christensen K,Xu C,Chai W,et al.Freeform inkjet printing of cellular structures with bifurcations.Biotechnol Bioeng.2015;112(5):1047-1055.

[9]Xu C,Chai W,Huang Y,et al.Scaffold-free inkjet printing of three-dimensional zigzag cellular tubes. Biotechnol Bioeng. 2015; 109(12):3152-3160.

[10]Ozbolat IT,Monika H.Current advances and future perspectives in extrusion-based bioprinting. Biomaterials.2016;76(37):321-343.

[11]Zhang Y,Yu Y,Ozbolat IT.Direct Bioprinting of Vessel-Like Tubular Microfluidic Channels.J Nanotechnol Eng Med.2013;4(2):21001.

[12]Massa S,Sakr MA,Seo J,et al. Bioprinted 3D vascularized tissue model for drug toxicity analysis. Biomicrofluidics.2017;11(4):44109.

[13]Zhang Y,Yu Y,Chen H,et al.Characterization of printable cellular micro-fluidic channels for tissue engineering.Biofabrication.2013;5(2): 25004.

[14]Luo Y,Lode A,Gelinsky M.Direct Plotting of Three‐Dimensional Hollow Fiber Scaffolds Based on Concentrated Alginate Pastes for Tissue Engineering. Adv Healthc Mater.2013;2(6):777-783.

[15]Cheng Y,Zheng F,Lu J,et al.Bioinspired multicompartmental microfibers from microfluidics. Adv Mater.2014;26(30):5184-5190.

[16]Gao Q,Liu Z,Lin Z,et al.3D Bioprinting of Vessel-like Structures with Multi-level Fluidic Channels.Acs Biomater Sci Eng.2017;3(3). DOI: 10.1021/acsbiomaterials.6b00643

[17]Onoe H,Okitsu T,Itou A, et al. Metre-long cell-laden microfibres exhibit tissue morphologies and functions.Nat Mater.2013;12(6):584.

[18]Rowley JA,Madlambayan G,Mooney DJ.Alginate hydrogels as synthetic extracellular matrix materials.Biomaterials.1999;20(1):45-53.

[19]Wang S, Zhang Y, Yin G, et al. Electrospun polylactide/silk fibroin– gelatin composite tubular scaffolds for small‐diameter tissue engineering blood vessels.J Appl Polym Sci. 2010;113(4): 2675-2682.

[20]Wang SD,Zhang YZ,Yin GB,et al.Fabrication of a composite vascular scaffold using electrospinning technology.MaterSci Eng C.2010;30(5): 670-676.

[21]Rodriguez MJ,Brown J,Giordano J,et al.Silk based bioinks for soft tissue reconstruction using 3-dimensional (3D) printing with in vitro and in vivo assessments. Biomaterials 2017;117:105-115.

[22]Chawla S,Midha S,Sharma A,et al.Silk‐Based Bioinks for 3D Bioprinting. Adv Healthc Mater. 2018;7(8):e1701204.

[23]Unger RE,Wolf M,Peters K,et al.Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering.Biomaterials. 2004;25(6):1069-1075.

[24]Liu H,Xu GW,Wang YF,et al.Composite scaffolds of nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell-based bone regeneration via the interleukin 1 alpha autocrine/paracrine signaling loop.Biomaterials. 2015;49:103-112.

[25]Zhou J, Cao C, Ma X, et al.In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds.Polym Degrad Stabil. 2010;95(9):1679-1685.

[26]Kweon HY, Yeo JH, Lee KG, et al. Effects of poloxamer on the gelation of silk sericin. Macromol Rapid Commun. 2000;21(18):1302-1305

[27]Kimsey RB,Spielman A.Motility of Lyme disease spirochetes in fluids as viscous as the extracellular matrix.J Infect Dis.1990;162(5):1205-1208.

[28]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.Biofabrication.2015;7(4):45002.

[29]Jia W,Gungor-Ozkerim PS,Zhang YS,et al.Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.Biomaterials. 2016; 106:58-68.

[30]王秀娟,张坤生,任云霞,等.海藻酸钠凝胶特性的研究[J].食品工业科技,2008, 29(2):259-262.

[31]Wang L,Xu M,Zhang L,et al. Automated quantitative assessment of three-dimensional bioprinted hydrogel scaffolds using optical coherence tomography.Biomed Opt Express.2016;7(3):894-910.

[32]Wang L,Xu ME, Luo L,et al.Iterative feedback bio-printing-derived cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability.Sci Rep.2018;8(1):2802.

[33]罗会涛,赵婧,范兴平,等.不同类型多孔结构生物材料支架制备及其性能优化[J].中国材料进展, 2012,31(5):30-39.

[34]Armstrong JP,Burke M, Carter BM, et al.3D Bioprinting Using a Templated Porous Bioink.Adv Healthc Mater. 2016;5(14):1724-1730.

[35]Kantlehner M,Schaffner P,Finsinger D,et al.Surface Coating with Cyclic RGD Peptides Stimulates Osteoblast Adhesion and Proliferation as well as Bone Formation.Chembiochem.2015;1(2):107-114.

[36]Ishaug-Riley SL,Crane-Kruger GM,Yaszemski MJ,et al. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers.Biomaterials. 1998;19(15):1405.

[37]Mikos AG,Lyman MD,Freed LE,et al.Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture.Biomaterials. 1994;15(1):55-58.

[38]Gao Q,He Y,Fu JZ,et al.Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery.Biomaterials. 2015;61: 203-215.