3D Printing of Layered Gradient Pore Structure of Brain-like Tissue
The pathological research and drug development of brain diseases require appropriate brain models. Given the complex, layered structure of the cerebral cortex, as well as the constraints on the medical ethics and the inaccuracy of animal models, it is necessary to construct a brain-like model in vitro. In this study, we designed and built integrated three-dimensional (3D) printing equipment for cell printing/culture, which can guarantee cell viability in the printing process and provide the equipment foundation for manufacturing the layered structures with gradient distribution of pore size. Based on this printing equipment, to achieve the purpose of printing the layered structures with multiple materials, we conducted research on the performance of bio-inks with different compositions and optimized the printing process. By extruding and stacking materials, we can print the layered structure with the uniform distribution of cells and the gradient distribution of pore sizes. Finally, we can accurately print a structure with 30 layers. The line width (resolution) of the printed monolayer structure was about 478 μm, the forming accuracy can reach 97.24%, and the viability of cells in the printed structure is as high as 94.5%.
1. Muming Pu BX, 2016, Nao Ke Xue Yu Lei Nao Yan Jiu Gai Shu. [Brain Science and Brain-Inspired Intelligence Technology]. J Chin Acad Sci, 31:725–36.
2. Xiong Y, Mahmood A, Chopp M, 2013, Animal Models of Traumatic Brain Injury. Nat Rev Neurosci, 14:128–42. https://doi.org/10.1038/nrn3407
3. Huh D, Hamilton GA, Ingber DE, 2011, From 3D Cell Culture to Organs-on-Chips. Trends Cell Biol, 21:745–54. https://doi.org/10.1016/j.tcb.2011.09.005
4. Imamura Y, Mukohara T, Shimono Y, et al., 2015, Comparison of 2D- and 3D-Culture Models as Drug-testing Platforms in Breast Cancer. Oncol Rep, 33:1837–43. https://doi.org/10.3892/or.2015.3767
5. Tian XF, Heng BC, Ge Z, et al., 2008, Comparison of Osteogenesis of Human Embryonic Stem Cells within 2D and 3D Culture systems. Scand J Clin Lab Invest, 68:58–6. https://doi.org/10.1080/00365510701466416
6. Zhang D, Pekkanen-Mattila M, Shahsavani M, et al., 2014, A 3D Alzheimer’s Disease Culture Model and the Induction of P21-Activated Kinase Mediated Sensing in iPSC Derived Neurons. Biomaterials, 35:1420–8. https://doi.org/10.1016/j.biomaterials.2013.11.028
7. Ng WL, Chua CK, Shen YF, 2019, Print Me An Organ! Why We Are Not There Yet. Prog Polym Sci, 97:101145. https://doi.org/10.1016/j.progpolymsci.2019.101145
8. Ozbolat IT, Hospodiuk M, 2016, Current Advances and Future Perspectives in Extrusion-based Bioprinting. Biomaterials, 76:321–43. https://doi.org/10.1016/j.biomaterials.2015.10.076
9. Zhuang P, Ng WL, An J, et al., 2019, Layer-by-layer Ultraviolet Assisted Extrusion-based (UAE) Bioprinting of Hydrogel Constructs with High Aspect Ratio for Soft Tissue Engineering Applications. PLoS One, 14:e0216776. https://doi.org/10.1371/journal.pone.0216776
10. Melchels FP, Feijen J, Grijpma DW, 2010, A Review on Stereolithography and its Applications in Biomedical Engineering. Biomaterials, 31:6121–30. https://doi.org/10.1016/j.biomaterials.2010.04.050
11. Saunders RE, Derby B, 2014, Inkjet Printing Biomaterials for Tissue Engineering: Bioprinting. Int Mater Rev, 59:430–48. https://doi.org/10.1179/1743280414y.0000000040
12. Koch LG, Unger C, Chichkov B, 2013, Laser Assisted Cell Printing. Curr Pharm Biotechnol, 14:91–7.
13. Ng WL, Lee JM, Yeong WY, et al., 2017, Microvalve-based Bioprinting-process, Bio-inks and Applications. Biomater Sci, 5:632–47. https://doi.org/10.1039/c6bm00861e
14. Ng WL, Goh MH, Yeong WY, et al., 2018, Applying Macromolecular Crowding to 3D Bioprinting: Fabrication of 3D Hierarchical Porous Collagen-based Hydrogel Constructs. Biomater Sci, 6:562–574. https://doi.org/10.1039/c7bm01015j
15. Tobin DJ, 2006, Biochemistry of Human Skin--our Brain on the Outside. Chem Soc Rev, 35:52–67. https://doi.org/10.1039/b505793k
16. Ng WL, Wang S, Yeong WY, et al., 2016, Skin Bioprinting: Impending Reality or Fantasy? Trends Biotechnol, 34:689–99. https://doi.org/10.1016/j.tibtech.2016.04.006
17. Ng WL, Qi JT, Yeong WY, et al., 2018, Proof-of-concept: 3D Bioprinting of Pigmented Human Skin Constructs. Biofabrication, 10:025005. https://doi.org/10.1088/1758-5090/aa9e1e
18. Meek KM, Knupp C, 2015, Corneal Structure and Transparency. Prog Retin Eye Res, 49:1–16.
19. Ferreira AM, Gentile P, Chiono V, et al., 2012, Collagen for Bone Tissue Regeneration. Acta Biomater, 8:3191–200. https://doi.org/10.1016/j.actbio.2012.06.014
20. Yao HB, Fang HY, Wang XH, et al., 2011, Hierarchical Assembly of Micro-/Nano-building Blocks: Bio-inspired Rigid Structural Functional Materials. Chem Soc Rev, 40:3764–85. https://doi.org/10.1039/c0cs00121j
21. Yang XY, Chen LH, Li Y, et al., 2017, Hierarchically Porous Materials: Synthesis Strategies and Structure Design. Chem Soc Rev, 46:481–558. https://doi.org/10.1039/c6cs00829a
22. Levingstone TJ, Matsiko A, Dickson GR, et al., 2014, A Biomimetic Multi-layered Collagen-based Scaffold for Osteochondral Repair. Acta Biomater, 10:1996–2004. https://doi.org/10.1016/j.actbio.2014.01.005
23. Tu DD, Chung YG, Gil ES, et al., 2013, Bladder Tissue Regeneration Using Acellular Bi-layer Silk Scaffolds in a Large Animal Model of Augmentation Cystoplasty. Biomaterials, 34:8681–9. https://doi.org/10.1016/j.biomaterials.2013.08.001
24. Xu R, Luo G, Xia H, et al., 2015, Novel Bilayer Wound Dressing Composed of Silicone Rubber with Particular Micropores Enhanced Wound Re-Epithelialization and Contraction. Biomaterials, 40:1–11. https://doi.org/10.1016/j.biomaterials.2014.10.077
25. Chung EJ, Ju HW, Park HJ, et al., 2015, Three-layered Scaffolds for Artificial Esophagus Using Poly (Varepsiloncaprolactone) Nanofibers and Silk Fibroin: An Experimental Study in a Rat Model. J Biomed Mater Res A, 103:2057–65. https://doi.org/10.1002/jbm.a.35347
26. Mi HY, Jing X, Yu E, et al., 2015, Fabrication of Triple layered Vascular Scaffolds by Combining Electrospinning, Braiding, and Thermally Induced Phase Separation. Mater Lett, 161:305–8. https://doi.org/10.1063/1.4937310
27. Wang LR, Thissen H, Jane A, et al., 2012, Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients. Adv Funct Mater, 22:3414–23. https://doi.org/10.1002/adfm.201200447
28. Wu C H, Lee F K, Kumar SS, et al., 2012, The Isolation and Differentiation of Human Adipose-derived Stem Cells Using Membrane Filtration. Biomaterials, 33:8228–39. https://doi.org/10.1016/j.biomaterials.2012.08.027
29. Nam H, Jeong HJ, Jo Y, et al., 2020, Multi-layered Freeform 3D Cell-printed Tubular Construct with Decellularized Inner and Outer Esophageal Tissue-derived Bioinks. Sci Rep, 10:7255. https://doi.org/10.1038/s41598-020-64049-6
30. Sun Y, You Y, Jiang W, et al., 2020, 3D Bioprinting Dual factor Releasing and Gradient-structured Constructs Ready to Implant for Anisotropic Cartilage Regeneration. Sci Adv, 6:eaay1422. https://doi.org/10.1126/sciadv.aay1422
31. Xu L, Varkey M, Jorgensen A, et al., 2020, Bioprinting Small Diameter Blood Vessel Constructs with an Endothelial and Smooth Muscle Cell Bilayer in a Single Step. Biofabrication, 12:045012. https://doi.org/10.1088/1758-5090/aba2b6
32. Kato-Negishi M, Morimoto Y, Onoe H, et al., 2013, Millimetersized Neural Building Blocks for 3D Heterogeneous Neural Network Assembly. Adv Healthc Mater, 2:1564–70. https://doi.org/10.1002/adhm.201300052
33. Sundararaghavan HG, Monteiro GA, Firestein BL, et al., 2009, Neurite Growth in 3D Collagen Gels with Gradients of Mechanical Properties. Biotechnol Bioeng, 102:632–43. https://doi.org/10.1002/bit.22074
34. NjNbRU, 2008, Microfluidic Generation of Biomaterial Gradients for Control of Neurite Outgrowth. Graduate School New Brunswick Electronic Theses and Dissertations.
35. Hopkins AM, DeSimone E, Chwalek K, et al., 2015, 3D In Vitro Modeling of the Central Nervous System. Prog Neurobiol, 125:1–25.
36. Bai H, Wang D, Delattre B, et al., 2015, Biomimetic Gradient Scaffold from Ice-templating for Self-seeding of Cells with Capillary Effect. Acta Biomater, 20:113–119. https://doi.org/10.1016/j.actbio.2015.04.007
37. Rnjak-Kovacina J, Wise SG, Li Z, et al., 2011, Tailoring the Porosity and Pore Size of Electrospun Synthetic Human Elastin Scaffolds for Dermal Tissue Engineering. Biomaterials, 32:6729–36. https://doi.org/10.1016/j.biomaterials.2011.05.065
38. Murphy CM, Haugh MG, O’Brien FJ, 2010, The Effect of Mean Pore Size on Cell Attachment, Proliferation and Migration in Collagen-glycosaminoglycan Scaffolds for Bone Tissue Engineering. Biomaterials, 31:461–6. https://doi.org/10.1016/j.biomaterials.2009.09.063
39. Lu H, Kawazoe N, Kitajima T, et al., 2012, Spatial Immobilization of Bone Morphogenetic Protein-4 in a Collagen-PLGA Hybrid Scaffold for Enhanced Osteoinductivity. Biomaterials, 33:6140–6. https://doi.org/10.1016/j.biomaterials.2012.05.038
40. Engelmayr GC Jr., Cheng M, Bettinger CJ, et al., 2008, Accordion-like Honeycombs for Tissue Engineering of Cardiac Anisotropy. Nat Mater, 7:1003–10. https://doi.org/10.1038/nmat2316
41. Pham QP, Shrma U, Mikos AG, 2006, Electrospun Poly(Ecaprolactone) Microfiber and Multilayer Nanofiber/Microfiber Scaffolds: Characterization of Scaffolds and Measurement of Cellular Infiltration. Biomacromolecules, 7:2796–2805. https://doi.org/10.1021/bm060680j
42. Chwalek K, Tang-Schomer MD, Omenetto FG, et al., 2015, In Vitro Bioengineered Model of Cortical Brain Tissue. Nat Protoc, 10:1362–73. https://doi.org/10.1038/nprot.2015.091
43. Odawara A, Gotoh M, Suzuki I, 2013, A Three-dimensional Neuronal Culture Technique that Controls the Direction of Neurite Elongation and the Position of Soma to Mimic the Layered Structure of the Brain. RSC Adv, 3:23620. https://doi.org/10.1039/c3ra44757j
44. Lee W, Pinckney J, Lee V, et al., 2009, Three-dimensional Bioprinting of Rat Embryonic Neural Cells. Neuroreport, 20:798–803. https://doi.org/10.1097/wnr.0b013e32832b8be4
45. Gu Q, Tomaskovic-Crook E, Lozano R, et al., 2016, Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells. Adv Healthc Mater, 5:1429–38. https://doi.org/10.1002/adhm.201670060
46. Lozano R, Stevens L, Thompson BC, et al., 2015, 3D Printing of Layered Brain-like Structures Using Peptide Modified Gellan Gum Substrates. Biomaterials, 67:264–73. https://doi.org/10.1016/j.biomaterials.2015.07.022
47. Gu ET, Wallace GG, Crook JM, 2017, Engineering Human Neural Tissue by 3D Bioprinting. In: Biomaterials for Tissue Engineering. Methods in Molecular Biology. p129–38. https://doi.org/10.1007/978-1-4939-7741-3_10
48. Vijayavenkataraman S, Vialli N, Fuh JY, et al., 2019, Conductive Collagen/Polypyrrole-b-polycaprolactone Hydrogel for Bioprinting of Neural Tissue Constructs. Int J Bioprint, 5:229. https://doi.org/10.18063/ijb.v5i2.1.229
49. Engler AJ, Sen S, Sweeney HL, et al., 2006, Matrix Elasticity Directs Stem Cell Lineage Specification. Cell, 126:677–89. https://doi.org/10.1016/j.cell.2006.06.044
50. Fischer T, Hayn A, Mierke CT, 2019, Fast and Reliable Advanced Two-step Pore-size Analysis of Biomimetic 3D Extracellular Matrix Scaffolds. Sci Rep, 9:8352. https://doi.org/10.1038/s41598-019-44764-5