AccScience Publishing / IJB / Volume 10 / Issue 3 / DOI: 10.36922/ijb.2219
RESEARCH ARTICLE

Enhancing cell proliferation in three-dimensional hydrogel scaffolds using digital light processing bioprinting technology

Yejin Choi1 Jeong Wook Seo1,2 Goo Jang3 Woo Kyung Jung2 Yong Ho Park2,4 Hojae Bae1,5*
Show Less
1 Department of Stem Cell and Regenerative Biotechnology, KU Convergence Science and Technology Institute, Konkuk University, Seoul, Republic of Korea
2 NoAH Biotech Co., Ltd., Suwon-si, Gyeonggi-do, Republic of Korea
3 Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Korea
4 Department of Microbiology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea
5 Institute of Advanced Regenerative Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, Republic of Korea
IJB 2024, 10(3), 2219 https://doi.org/10.36922/ijb.2219
Submitted: 9 November 2023 | Accepted: 1 February 2024 | Published: 28 March 2024
(This article belongs to the Special Issue Light-based bioprinted scaffolds for tissue engineering)
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Three-dimensional (3D) bioprinting is gradually emerging as a popular technique driving as a new paradigm in tissue engineering. Enhancing cell proliferation and engraftment within volumetric 3D-bioprinted scaffolds is a key challenge in its implementation. However, basic exploratory studies on cell proliferation enhancement in 3D-bioprinted scaffolds using digital light processing (DLP) technology are still lacking. Traditionally, microchannels in scaffolds have been regarded as non-functional, empty spaces. In this paper, however, we propose that microchannels implanted in DLP-bioprinted scaffolds can provide space for cell proliferation, giving a new definition to microchannel function. To this end, we used fish gelatin methacrylate (F-GelMA) as a bioink with photocurable properties, followed by functional evaluation and optimization through rheological analysis. The morphology of DLP-printed scaffolds using the bioink was analyzed, and their biocompatibility was demonstrated through cell viability analysis. Microchannels of three different sizes were implanted to facilitate oxygenation, nutrient delivery, and media flow by addressing structural barriers identified via morphological analysis. Cell viability and proliferation rates in outer and inner microchannels were then comparatively analyzed. During the long-term culture period (about 5 weeks), the differences in proliferation rates due to changes in the media flow environment were assessed. The results demonstrated that cell survival, growth, and proliferation were significantly enhanced within the DLP-printed scaffolds in which the cells were encapsulated. This approach lends itself useful for basic exploratory study utilizing 3D culture technology in the realms of regenerative medicine and tissue engineering, where effective cell proliferation relative to the same volume is required.

 

Keywords
Tissue engineering
Digital light processing
Three-dimensional printing
Hydrogel scaffold
Cell proliferation
Microchannel
Funding
This work was supported by the Creative and Challenging Convergence Model Development Program (RS-2023- 00232550) from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (iPET). The authors are grateful for the financial support from the National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2018R1D1A1B05047274) and NoAH Biotech Co., Ltd. Korea.
Conflict of interest
The authors declare no conflicts of interest.
References
  1. Cohan M. From petri dish to dinner plate: this is the world’s first 3D-printed, cultivated fish fillet. CNN; 2023. https://edition.cnn.com/travel/article/steakholder-foods- 3d-printed-cultivated-fish-fillet-spc-intl/index.html
  2. Listek V. Aleph farms’ new cultured steak to join the cultured meat race. 3D print.com; 2023. https://3dprint.com/299484/aleph-farms-new-3d-printed-steak-to-join-the-cultured-meat-race/
  3. Marr B. The future of food: amazing lab grown and 3D printed meat and fish. Forbes; 2019. https://www.forbes.com/sites/bernardmarr/2019/06/28/ the-future-of-food-amazing-lab-grown-and-3d-printed-meat-and-fish/?sh=6fdf360446f6
  4. Reiley L. Raising the steaks: first 3-D-printed rib-eye is unveiled. The Washington Post; 2021. https://www.washingtonpost.com/business/2021/02/09/3d-printed-ribeye-steak-usda-fda/
  5. Ianovici I, Zagury Y, Redenski I, Lavon N, Levenberg S. 3D-printable plant protein-enriched scaffolds for cultivated meat development. Biomaterials. 2022;284:121487. doi: 10.1016/j.biomaterials.2022.121487
  6. Jeong D, Seo JW, Lee H-G, Jung WK, Park YH, Bae H. Efficient myogenic/adipogenic transdifferentiation of bovine fibroblasts in a 3D bioprinting system for steak- type cultured meat production. Adv Sci. 2022;9(31): 2202877. doi: 10.1002/advs.202202877
  7. Su L, Jing L, Zeng X, et al. 3D-printed prolamin scaffolds for cell-based meat culture. Adv Mater. 2023;35(2):2207397. doi: 10.1002/adma.202207397
  8. Nam HK, Kang TW, Kim I-W, Choi R-Y, Kim HW, Park HJ. Physicochemical properties of cricket (Gryllus bimaculatus) gel fraction with soy protein isolate for 3D printing-based meat analogue. Food Biosci. 2023;53:102772. doi: 10.1016/j.fbio.2023.102772
  9. Mani MP, Sadia M, Jaganathan SK, et al. A review on 3D printing in tissue engineering applications. J Polym Eng. 2022;42(3):243-265. doi: 10.1515/polyeng-2021-0059
  10. Samadi A, Moammeri A, Pourmadadi M, et al. Cell encapsulation and 3D bioprinting for therapeutic cell transplantation. ACS Biomater Sci Eng. 2023;9(4):1862-1890. doi: 10.1021/acsbiomaterials.2c01183
  11. Wu Y, Su H, Li M, Xing H. Digital light processing-based multi-material bioprinting: processes, applications, and perspectives. J Biomed Mater Res A. 2023;111(4):527-542. doi: 10.1002/jbm.a.37473
  12. Li W, Wang M, Ma H, Chapa-Villarreal FA, Lobo AO, Zhang YS. Stereolithography apparatus and digital light processing-based 3D bioprinting for tissue fabrication. Iscience. 2023;26(2):106039. doi: 10.1016/j.isci.2023.106039
  13. Xing F, Xu J, Yu P, et al. Recent advances in biofabrication strategies based on bioprinting for vascularized tissue repair and regeneration. Mater Des. 2023;111885. doi: 10.1016/j.matdes.2023.111885
  14. Song P, Li M, Zhang B, et al. DLP fabricating of precision GelMA/HAp porous composite scaffold for bone tissue engineering application. Compos Part B: Eng. 2022;244:110163. doi: 10.1016/j.compositesb.2022.110163
  15. Ye W, Li H, Yu K, et al. 3D printing of gelatin methacrylate-based nerve guidance conduits with multiple channels. Mater Des. 2020;192:108757. doi: 10.1016/j.matdes.2020.108757
  16. Peng M, Zhao Q, Wang M, Du X. Reconfigurable scaffolds for adaptive tissue regeneration. Nanoscale. 2023;15(13): 6105-6120. doi: 10.1039/D3NR00281K
  17. Pham CH, Zuo Y, Chen Y, Tran NM, Nguyen DT, Dang TT. Waffle‐inspired hydrogel‐based macrodevice for spatially controlled distribution of encapsulated therapeutic microtissues and pro‐angiogenic endothelial cells. Bioeng Transl Med. 2023;8(3):e10495. doi: 10.1002/btm2.10495 
  18. Malda J, Rouwkema J, Martens DE, et al. Oxygen gradients in tissue-engineered Pegt/Pbt cartilaginous constructs: measurement and modeling. Biotechnol Bioeng. 2004;86(1):9-18. doi: 10.1002/bit.20038
  19. Laschke MW, Harder Y, Amon M, et al. Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng. 2006;12(8):2093-2104. doi: 10.1089/ten.2006.12.2093
  20. Grebenyuk S, Abdel Fattah AR, Kumar M, et al. Large-scale perfused tissues via synthetic 3D soft microfluidics. Nat Commun. 2023;14(1):193. doi: 10.1038/s41467-022-35619-1
  21. Zohar B, Debbi L, Machour M, et al. A micro-channel array in a tissue engineered vessel graft guides vascular morphogenesis for anastomosis with self-assembled vascular networks. Acta Biomater. 2022;163:182-193. doi: 10.1016/j.actbio.2022.05.026
  22. Luo Y, Zhang T, Lin X. 3D printed hydrogel scaffolds with macro pores and interconnected microchannel networks for tissue engineering vascularization. Chem Eng J. 2022;430:132926. doi: 10.1016/j.cej.2021.132926
  23. Tang F, Manz XD, Bongers A, et al. Microchannels are an architectural cue that promotes integration and vascularization of silk biomaterials in vivo. ACS Biomater Sci Eng. 2020;6(3):1476-1486. doi: 10.1021/acsbiomaterials.9b01624
  24. Lim KS, Baptista M, Moon S, Woodfield TBF, Rnjak- Kovacina J. Microchannels in development, survival, and vascularisation of tissue analogues for regenerative medicine. Trends Biotechnol. 2019;37(11):1189-1201. doi: 10.1016/j.tibtech.2019.04.004
  25. Miller JS, Stevens KR, Yang MT, et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater. 2012;11(9):768-774. doi: 10.1038/nmat3357
  26. Mainardi VL, Rubert M, Sabato C, et al. Culture of 3D bioprinted bone constructs requires an increased fluid dynamic stimulation. Acta Biomater. 2022;153:374-385. doi: 10.1016/j.actbio.2022.09.011
  27. Homan KA, Gupta N, Kroll KT, et al. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat Methods. 2019;16(3):255-262. doi: 10.1038/s41592-019-0325-y
  28. Tang MD, Golden AP, Tien J. Fabrication of collagen gels that contain patterned, micrometer-scale cavities. Adv Mater. 2004;16(15):1345-1348. doi: 10.1002/adma.200400766
  29. Rouwkema J, Rivron NC, van Blitterswijk CA. Vascularization in tissue engineering. Trends Biotechnol. 2008;26(8):434-441. doi: 10.1016/j.tibtech.2008.04.009
  30. Xia P, Luo Y. Vascularization in tissue engineering: the architecture cues of pores in scaffolds. J Biomed Mater Res Part B App Biomater. 2022;110(5):1206-1214. doi: 10.1002/jbm.b.34979
  31. Seo JW, Kim GM, Choi Y, Cha JM, Bae H. Improving printability of digital-light-processing 3D bioprinting via photoabsorber pigment adjustment. Int J Mol Sci. 2022;23(10):5428. doi: 10.3390/ijms23105428
  32. Chavez T, Gerecht S. Engineering of the microenvironment to accelerate vascular regeneration. Trends Mol Med. 2023;29(1):35-47. doi: 10.1016/j.molmed.2022.10.005
  33. de Souza A, Martignago CCS, Santo GdE, et al. 3D printed wound constructs for skin tissue engineering: a systematic review in experimental animal models. J Biomed Mater Res Part B Appl Biomater. 2023;111(7):1419-1433. doi: 10.1002/jbm.b.35237
  34. Jodat YA, Zhang T, Tanoury Z, et al. hiPSC-Derived 3D Bioprinted Skeletal Muscle Tissue Implants Regenerate Skeletal Muscle Following Volumetric Muscle Loss. Research Square Publications; 2021. doi: 10.21203/rs.3.rs-146091/v1 
  35. Ciolacu DE, Nicu R, Ciolacu F. Natural polymers in heart valve tissue engineering: strategies, advances and challenges. Biomedicines. 2022;10(5):1095. doi: 10.3390/biomedicines10051095
  36. Yan J, Li Z, Guo J, Liu S, Guo J. Organ-on-a-chip: a new tool for in vitro research. Biosens Bioelectron. 2022;216:114626. doi: 10.1016/j.bios.2022.114626
  37. Jakab K, Marga F, Kaesser R, et al. Non-medical applications of tissue engineering: biofabrication of a leather-like material. Mater Today Sustain. 2019;5:100018. doi: 10.1016/j.mtsust.2019.100018
  38. Decante G, Costa JB, Silva-Correia J, Collins MN, Reis RL, Oliveira JM. Engineering bioinks for 3D bioprinting. Biofabrication. 2021;13(3):032001. doi: 10.1088/1758-5090/abec2c
  39. Van den Steen PE, Dubois B, Nelissen I, Rudd PM, Dwek RA, Opdenakker G. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Crit Rev Biochem Mol Biol. 2002;37(6):375-536. doi: 10.1080/10409230290771546
  40. Nichol JW, Koshy ST, Bae H, Hwang CM, Yamanlar S, Khademhosseini A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. 2010;31(21): 5536-5544. doi: 10.1016/j.biomaterials.2010.03.064
  41. Yoon HJ, Shin SR, Cha JM, et al. Cold water fish gelatin methacryloyl hydrogel for tissue engineering application. PLOS ONE. 2016;11(10):e0163902. doi: 10.1371/journal.pone.0163902
  42. Randhawa A, Dutta SD, Ganguly K, Patel DK, Patil TV, Lim K-T. Recent advances in 3D printing of photocurable polymers: types, mechanism, and tissue engineering application. Macromol Biosci. 2023;23(1):2200278. doi: 10.1002/mabi.202200278
  43. Huh J, Moon Y-W, Park J, Atala A, Yoo JJ, Lee SJ. Combinations of photoinitiator and UV absorber for cell-based digital light processing (DLP) bioprinting. Biofabrication. 2021;13(3):034103. doi: 10.1088/1758-5090/abfd7a
  44. Chen Y, Zhang J, Liu X, et al. Noninvasive in vivo 3D bioprinting. Sci Adv. 2020;6(23):eaba7406. doi: 10.1126/sciadv.aba7406
  45. Goodarzi Hosseinabadi H, Dogan E, Miri AK, Ionov L. Digital light processing bioprinting advances for microtissue models. ACS Biomater Sci Eng. 2022;8(4):1381-1395. doi: 10.1021/acsbiomaterials.1c01509
  46. Alberts B, Johnson A, Lewis J, et al. The extracellular matrix of animals. In: Molecular Biology of the Cell. 4th ed. New York: Garland Science; 2002.
  47. Lee RC, Ping J. Calcium antagonists retard extracellular matrix production in connective tissue equivalent. J Surg Res. 1990;49(5):463-466. doi: 10.1016/0022-4804(90)90197-A
  48. Schmid GJ, Kobayashi C, Sandell LJ, Ornitz DM. Fibroblast growth factor expression during skeletal fracture healing in mice. Dev Dyn. 2009;238(3):766-774. doi: 10.1002/dvdy.21882
  49. Ross R. The fibroblast and wound repair. Biol Rev. 1968;43(1):51-91. doi: 10.1111/j.1469-185X.1968.tb01109.x
  50. Darby IA, Hewitson TD. Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol. 2007;257:143-179. doi: 10.1016/S0074-7696(07)57004-X
  51. Seo JW, Moon JH, Jang G, et al. Cell-laden gelatin methacryloyl bioink for the fabrication of Z-stacked hydrogel scaffolds for tissue engineering. Polymers. 2020;12(12):3027. doi: 10.3390/polym12123027
  52. Leu Alexa R, Iovu H, Ghitman J, et al. 3D-printed gelatin methacryloyl-based scaffolds with potential application in tissue engineering. Polymers. 2021;13(5):727. doi: 10.3390/polym13050727
  53. Zu G, Meijer M, Mergel O, Zhang H, van Rijn P. 3D-printable hierarchical nanogel-GelMA composite hydrogel system. Polymers. 2021;13(15):2508. doi: 10.3390/polym13152508 
  54. Ma C, Choi J-B, Jang Y-S, et al. Mammalian and fish gelatin methacryloyl–alginate interpenetrating polymer network hydrogels for tissue engineering. ACS Omega. 2021;6(27):17433-17441. doi: 10.1021/acsomega.1c01806
  55. Garbern JC, Hoffman AS, Stayton PS. Injectable pH- and temperature-responsive poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers for delivery of angiogenic growth factors. Biomacromolecules. 2010;11(7): 1833-1839. doi: 10.1021/bm100318z
  56. Yu K, Zhang X, Sun Y, et al. Printability during projection-based 3D bioprinting. Bioact Mater. 2022;11:254-267. doi: 10.1016/j.bioactmat.2021.09.021
  57. Zeltinger J, Sherwood JK, Graham DA, Müeller R, Griffith LG. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng. 2001;7(5):557-572. doi: 10.1089/107632701753213183
  58. Rather JA, Akhter N, Ashraf QS, et al. A comprehensive review on gelatin: Understanding impact of the sources, extraction methods, and modifications on potential packaging applications. Food Pkg Shelf Life. 2022;34:100945. https://www.sciencedirect.com/science/article/pii/ S2214289422001375
  59. Andreazza R, Morales A, Pieniz S, Labidi J. Gelatin-based hydrogels: potential biomaterials for remediation. Polymers. 2023;15(4):1026. doi: 10.3390/polym15041026
  60. Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. 2020;7:33. doi: 10.3389/fmolb.2020.00033
  61. Wang Y, Huang X, Shen Y, et al. Direct writing alginate bioink inside pre-polymers of hydrogels to create patterned vascular networks. J Mater Sci. 2019;54(10):7883-7892. doi: 10.1007/s10853-019-03447-2
  62. Ying GL, Jiang N, Maharjan S, et al. Aqueous two-phase emulsion bioink-enabled 3D bioprinting of porous hydrogels. Adv Mater. 2018;30(50):e1805460. doi: 10.1002/adma.201805460
  63. Song YS, Lin RL, Montesano G, et al. Engineered 3D tissue models for cell-laden microfluidic channels. Anal Bioanal Chem. 2009;395(1):185-193. doi: 10.1007/s00216-009-2935-1
  64. Rnjak-Kovacina J, Wray LS, Golinski JM, Kaplan DL. Arrayed hollow channels in silk-based scaffolds provide functional outcomes for engineering critically sized tissue constructs. Adv Funct Mater. 2014;24(15):2188-2196. doi: 10.1002/adfm.201302901
  65. Wen N, Qian E, Kang Y. Effects of macro-/micro-channels on vascularization and immune response of tissue engineering scaffolds. Cells. 2021;10(6):1514. doi: 10.3390/cells10061514
  66. Xie Y, Hardouin P, Zhu Z, Tang T, Dai K, Lu J. Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size β-tricalcium phosphate scaffold. Tissue Eng. 2006;12(12):3535-3543. doi: 10.1089/ten.2006.12.3535
  67. Nii T, Makino K, Tabata Y. Influence of shaking culture on the biological functions of cell aggregates incorporating gelatin hydrogel microspheres. J Biosci Bioeng. 2019;128(5):606- 612. doi: 10.1016/j.jbiosc.2019.04.013
  68. Limraksasin P, Kosaka Y, Zhang M, et al. Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs. Sci Rep. 2020;10(1):14996. doi: 10.1038/s41598-020-72038-y
  69. Li C, Kuss M, Kong Y, et al. 3D printed hydrogels with aligned microchannels to guide neural stem cell migration. ACS Biomater Sci Eng. 2021;7(2):690-700. doi: 10.1021/acsbiomaterials.0c01619
  70. Han J, Park S, Kim JE, et al. Development of a scaffold-on-a-chip platform to evaluate cell infiltration and osteogenesis on the 3D-printed scaffold for bone regeneration. ACS Biomater Sci Eng. 2023;9(2):968-977. doi: 10.1021/acsbiomaterials.2c01367 
  71. Dhwaj A, Roy N, Jaiswar A, Prabhakar A, Verma D. 3D-printed impedance micropump for continuous perfusion of the sample and nutrient medium integrated with a liver-on-chip prototype. ACS Omega. 2022;7(45):40900-40910. doi: 10.1021/acsomega.2c03818
  72. Gao J, Li M, Cheng J, et al. 3D-printed GelMA/PEGDA/ F127DA scaffolds for bone regeneration. J Funct Biomater. 2023;14(2):96. doi: 10.3390/jfb14020096
  73. Ma H, Xing F, Yu P, et al. Integrated design and fabrication strategies based on bioprinting for skeletal muscle regeneration: current status and future perspectives. Mater Des. 2023;225:111591. doi: 10.1016/j.matdes.2023.111591
  74. Yu Q, Wang Q, Zhang L, et al. The applications of 3D printing in wound healing: the external delivery of stem cells and antibiosis. Adv Drug Del Rev. 2023;197:114823. doi: 10.1016/j.addr.2023.114823
Share
Back to top
International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing