傳統的體外二維培養具方便易觀察的優點，但二維模型往往無法模擬體內器官的立體結構和微環境。體外三維模型提供了一種新方法，可以彌補傳統 2D 培養和動物模型之間的差距。大腦是人類最複雜的器官之一，且神經退化性疾病已成為未來老年化社會的重要問題。水膠載體的開發有助於神經組織工程的應用；而三維體外大腦模型的建構將能有助於探討疾病問題、輔助藥物篩選、以及新療法的開發。三維生物列印是近年熱門的體外模型建構工具之一，但模擬大腦的三維生物列印仍有許多待突破的困境。為了能夠更真實模擬神經網絡在三維結構下生長的微環境，需要建構可以準確模擬適合神經細胞生長的微環境並貼近人體內生理環境組成的三維結構。本研究旨在開發適合神經生長環境的生物墨水、建構體外模擬3D大腦的微環境，並探討其應用於3D大腦列印的可行性。
為了開發可以應用於模擬真實大腦組織的三維列印仿生墨水，本研究萃取蠶絲蛋白並經經過GMA(Glycidyl Methacrylate)化學修飾改質為光交聯甲基丙烯醯化蠶絲蛋白 (Sil-MA)，並添加取自水果果皮的Pectin以製備系列Sil-MA-Pectin生物墨水。Sil-MA是一種具有高生物相容性與機械性質的水膠，而藉由添加不同濃度之Pectin可調整水膠的軟硬度並增加其可列印性。GMA對蠶絲蛋白的修飾可以通過在傅立葉變換紅外光譜 (FT-IR) 和核磁共振光譜 (H-NMR)中檢視GMA以及SF 的特徵相關峰，並證明GMA對蠶絲蛋白的修飾。此外，藉由量測Sil-MA-Pectin水膠的機械和流變性質，發現可以透過改變 Sil-MA 或Pectin的濃度來調整水膠的機械性質。隨著Sil-MA濃度的增加，水膠的壓縮模量會增加，但反之Pectin的加入越多，水膠會變得越來越軟。此系統可利用兩種交聯方式成膠，Sil-MA是透過光交聯成膠，Pectin則是透過離子交聯。Sil-MA-Pectin水膠的掃描電子顯微照片（SEM）顯示出水膠微觀結構，表現出適合細胞生長或延伸的多孔型態。細胞活性測試以及毒性測試證明系列Sil-MA-Pectin水膠均具有良好的生物相容性。為了探討此系列水膠應用於體外大腦模型建構與神經組織工程的可行性，接著將取自懷孕16天的母鼠胚胎的神經幹細胞球體包覆在系列Sil-MA-Pectin水膠中以觀察神經幹細胞在三維模型中的分化情形，並藉由染色結果進行量化分析。結果顯示，在未添加任何生長因子的環境中，神經幹細胞在系列Sil-MA-Pectin水膠均展現良好的分化性質，其中神經元的分化百分比均高於80%。而可列印性測試證實15%SilMA-0.5%Pectin具有最佳的可列印性。因此最後使用此生物墨水以擠出式3D列印機對包埋神經幹細胞的生物墨水進行列印，細胞死活與免疫染色結果均顯示，神經幹細胞通過列印噴嘴後仍然可以保持高活力狀態並發現Sil-MA-Pectin生物墨水依然展現出良好的分化性質，其中神經元的分化百分比均高於80%，成功建構出模擬大腦的3D體外模型。
本研究使用以甲基丙烯醯化蠶絲蛋白與果膠結合製備出的3D水膠模擬體外大腦微環境，能夠有效搭載神經幹細胞並促進神經幹細胞的分化與生長，未來可能開發出更複雜的3D體外大腦模型或建立其他疾病模型並應用於神經組織工程中。

Generally, conventional in vitro cell-based evaluations are implemented under two-dimensional culture (2D) conditions, however, Common 2D models easy to observe but lack the proper recapitulation of organ structure and environment. In vitro 3D model provides a new approach which bridges the gap between traditional 2D culture and animal model. Brain is one of the most complex organs in human beings, and neurodegenerative diseases have become an important issue in the future. The development of hydrogel carriers is conducive to the application of neural tissue engineering; and the construction of 3D in vitro brain models will help to explore disease problems, assist drug screening, and develop new therapies. 3D bioprinting is one of the popular in vitro model building tools in recent years, but there are still many difficulties to be overcome in 3D bioprinting simulating the brain. Therefore, it is necessary to create a platform consist of cell models that accurately simulate the cells microenvironment, along with flexibly prototyped cell handling structures that mimic the in vivo environment.
Hydrogels are promising biomedical materials because they resemble biological tissues with regard to their morphology, high degree of flexibility and water holding capacity.
To develop a 3D in vitro model that can mimic the real tissue, photo-crosslinkable acid silk fibroin (Sil-MA) and Pectin, an inexpensive and negatively charged polysaccharide that is found in the cell walls of terrestrial plants, were used to prepare the Sil-MA/Pectin hydrogels. Sil-MA is a hydrogel with high biocompatibility and mechanical properties. By adding different concentrations of Pectin, the hardness of the hydrogel can be adjusted and its printability can be increased.
Modification of Glycidyl Methacrylate (GMA) on silk fibroin was confirmed through identification of GMA-related peaks and Silk Fibroin(SF)-related peaks by Fourier-transform infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance spectroscopy (H-NMR). Besides, the mechanical and rheological properties of Sil-MA/Pectin hydrogel were determined and it is revealed that the mechanical properties were modulated by varying the Sil-MA or pectin contents, With the increase of Sil-MA concentration, the compressive modulus increase of the hydrogel were observed. In contrast, when more Pectin is added, the hydrogel become softer. There are two kinds of crosslinking mechanism were used in this study, Sil-MA is cross-linked by UV and Pectin is through ionic cross-linking. Scanning electron micrographs photographs of Sil-MA-Pectin hydrogels show that hydrogel demonstrated porous morphology which is suitable for cell growth, and cell viability test also proved that Sil-MA/Pectin hydrogel showed good biocompatibility.
In order to evaluate the feasibility of the application of the series of Silk fibroin based bioinks on neural engineering and the in vitro model, Neural stem cells (NSCs) spheroids obtained from pregnant ED 16 Wistar rat embryos were encapsulated in a series of Sil-MA-Pectin hydrogels to observe the differentiation of neural stem cells in a 3D model. In addition, the differentiation percentages of neuron/astrocyte were analyzed by immunostaining. The results showed that NSCs exhibited good differentiation properties in the series of Sil-MA-Pectin hydrogels, and the percentage of neuron differentiation was higher than 80%.
The printability test confirmed that 15% SilMA-0.5% Pectin showed the best printability. Therefore, this hydrogel was finally used on NSCs spheroid laden 3D bioprinting by an extrusion 3D printer. The results of Live&Dead and immunostaining all showed that the NSCs spheroids can still maintain a high vitality state after passing through the printing nozzle.
In this study, 3D printed used bio-inks prepared by Sil-MA and pectin was used to simulate the brain in vitro microenvironment, which can effectively carry NSCs and promote the differentiation and growth. It is considered that series of adjustable Sil-MA/pectin bio-inks provides the alternative on 3D bio-printing for in vitro biological tissue model development. In the future, it is possible to develop more complex 3D in vitro Brain models or other disease models and applied in neural tissue engineering.

1. Qi, Z., et al., A dual-drug enhanced injectable hydrogel incorporated with neural stem cells for combination therapy in spinal cord injury. Chemical Engineering Journal, 2022. 427: p. 130906.
2. Gage, F.H., Mammalian neural stem cells. Science, 2000. 287(5457): p. 1433-1438.
3. Ourednik, V., et al., Segregation of human neural stem cells in the developing primate forebrain. Science, 2001. 293(5536): p. 1820-1824.
4. Kornblum, H.I., Introduction to neural stem cells. Stroke, 2007. 38(2): p. 810-816.
5. Brushart, T.M., et al., Electrical stimulation promotes motoneuron regeneration without increasing its speed or conditioning the neuron. Journal of Neuroscience, 2002. 22(15): p. 6631-6638.
6. Lee, I.-C. and Y.-C. Wu, Facilitating neural stem/progenitor cell niche calibration for neural lineage differentiation by polyelectrolyte multilayer films. Colloids and Surfaces B: Biointerfaces, 2014. 121: p. 54-65.
7. Mahairaki, V., et al., Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro. Tissue Engineering Part A, 2011. 17(5-6): p. 855-863.
8. Keating, A., et al., Donor origin of the in vitro haematopoietic microenvironment after marrow transplantation in man. Nature, 1982. 298(5871): p. 280-283.
9. Kaplowitz, N., Idiosyncratic drug hepatotoxicity. Nature reviews Drug discovery, 2005. 4(6): p. 489-499.
10. Ma, X., et al., Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. Proceedings of the National Academy of Sciences, 2016. 113(8): p. 2206-2211.
11. Akay, M., et al., Drug screening of human GBM spheroids in brain cancer chip. Scientific reports, 2018. 8(1): p. 1-9.
12. Di Lullo, E. and A.R. Kriegstein, The use of brain organoids to investigate neural development and disease. Nature Reviews Neuroscience, 2017. 18(10): p. 573-584.
13. Ngo, T.B., et al., Three-Dimensional bioprinted hyaluronic acid hydrogel test beds for assessing neural cell responses to competitive growth stimuli. ACS Biomaterials Science & Engineering, 2020. 6(12): p. 6819-6830.
14. Ahn, S.I., et al., Microengineered human blood–brain barrier platform for understanding nanoparticle transport mechanisms. Nature communications, 2020. 11(1): p. 1-12.
15. Gebeyehu, A., et al., Polysaccharide hydrogel based 3D printed tumor models for chemotherapeutic drug screening. Scientific reports, 2021. 11(1): p. 1-15.
16. Cui, X., et al., Advances in extrusion 3D bioprinting: a focus on multicomponent hydrogel‐based bioinks. Advanced Healthcare Materials, 2020. 9(15): p. 1901648.
17. Ye, W., et al., 3D printing of gelatin methacrylate-based nerve guidance conduits with multiple channels. Materials & Design, 2020. 192: p. 108757.
18. Oliveira, H., et al., Extracellular matrix (ECM)-derived bioinks designed to foster vasculogenesis and neurite outgrowth: Characterization and bioprinting. Bioprinting, 2021. 22: p. e00134.
19. Ong, C.S., et al., 3D bioprinting using stem cells. Pediatric research, 2018. 83(1): p. 223-231.
20. Kim, S.H., et al., Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing. Nature communications, 2018. 9(1): p. 1-14.
21. Hong, H., et al., Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering. Biomaterials, 2020. 232: p. 119679.
22. Ajiteru, O., et al., A digital light processing 3D printed magnetic bioreactor system using silk magnetic bioink. Biofabrication, 2021. 13(3): p. 034102.
23. Liu, Y., et al., Semi-interpenetrating polymer network of hyaluronan and chitosan self-healing hydrogels for central nervous system repair. ACS Applied Materials & Interfaces, 2020. 12(36): p. 40108-40120.
24. Xiao, W., et al., Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels. Acta biomaterialia, 2011. 7(6): p. 2384-2393.
25. Dyakonov, T., et al., Design and characterization of a silk-fibroin-based drug delivery platform using naproxen as a model drug. Journal of Drug Delivery, 2012. 2012.
26. Cheng, Y., et al., On the strength of β-sheet crystallites of Bombyx mori silk fibroin. Journal of the Royal Society Interface, 2014. 11(96): p. 20140305.
27. Li, X., et al., Water-stable silk fibroin nerve conduits with tunable degradation prepared by a mild freezing-induced assembly. Polymer Degradation and Stability, 2019. 164: p. 61-68.
28. Ma, T., et al., Rheological behavior and particle alignment of cellulose nanocrystal and its composite hydrogels during 3D printing. Carbohydrate Polymers, 2021. 253: p. 117217.
29. Keppler, F., et al., Methane emissions from terrestrial plants under aerobic conditions. Nature, 2006. 439(7073): p. 187-191.
30. Mehrali, M., et al., Pectin methacrylate (PEMA) and gelatin-based hydrogels for cell delivery: converting waste materials into biomaterials. ACS applied materials & interfaces, 2019. 11(13): p. 12283-12297.
31. Vandenburgh, H.H. and P. Karlisch, Longitudinal growth of skeletal myotubes in vitro in a new horizontal mechanical cell stimulator. In vitro cellular & developmental biology, 1989. 25(7): p. 607-616.
32. Kim, S.H., et al., 4D-bioprinted silk hydrogels for tissue engineering. Biomaterials, 2020. 260: p. 120281.
33. Richardson, M. and B. Haylock, Designer/maker: the rise of additive manufacturing, domestic-scale production and the possible implications for the automotive industry. Computer-Aided Design & Applications PACE, 2012. 2: p. 33-48.
34. Garreta, E., et al., Tissue engineering by decellularization and 3D bioprinting. Materials Today, 2017. 20(4): p. 166-178.
35. Li, C., et al., 3D printed hydrogels with aligned microchannels to guide neural stem cell migration. ACS Biomaterials Science & Engineering, 2021. 7(2): p. 690-700.
36. Hamid, O.A., et al., 3D bioprinting of a stem cell-laden, multi-material tubular composite: An approach for spinal cord repair. Materials Science and Engineering: C, 2021. 120: p. 111707.
37. Vancauwenberghe, V., et al., Development of a coaxial extrusion deposition for 3D printing of customizable pectin-based food simulant. Journal of Food Engineering, 2018. 225: p. 42-52.
38. Fan, L., et al., Directing induced pluripotent stem cell derived neural stem cell fate with a three-dimensional biomimetic hydrogel for spinal cord injury repair. ACS applied materials & interfaces, 2018. 10(21): p. 17742-17755.