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Polyvinylpyrrolidone-based bioink: influence of bioink properties on printing performance and cell proliferation during inkjet-based bioprinting

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Abstract

Among the different bioprinting techniques, the drop-on-demand (DOD) jetting-based bioprinting approach facilitates contactless deposition of pico/nanoliter droplets of materials and cells for optimal cell‒matrix and cell‒cell interactions. Although bioinks play a critical role in the bioprinting process, there is a poor understanding of the influence of bioink properties on printing performance (such as filament elongation, formation of satellite droplets, and droplet splashing) and cell health (cell viability and proliferation) during the DOD jetting-based bioprinting process. An inert polyvinylpyrrolidone (PVP360, molecular weight=360 kDa) polymer was used in this study to manipulate the physical properties of the bioinks and investigate the influence of bioink properties on printing performance and cell health. Our experimental results showed that a higher bioink viscoelasticity helps to stabilize droplet filaments before rupturing from the nozzle orifice. The highly stretched droplet filament resulted in the formation of highly aligned “satellite droplets,” which minimized the displacement of the satellite droplets away from the predefined positions. Next, a significant increase in the bioink viscosity facilitated droplet deposition on the wetted substrate surface in the absence of splashing and significantly improved the accuracy of the deposited main droplet. Further analysis showed that cell-laden bioinks with higher viscosity exhibited higher measured average cell viability (%), as the presence of polymer within the printed droplets provides an additional cushioning effect (higher energy dissipation) for the encapsulated cells during droplet impact on the substrate surface, improves the measured average cell viability even at higher droplet impact velocity and retains the proliferation capability of the printed cells. Understanding the influence of bioink properties (e.g., bioink viscoelasticity and viscosity) on printing performance and cell proliferation is important for the formulation of new bioinks, and we have demonstrated precise DOD deposition of living cells and fabrication of tunable cell spheroids (nL‒µL range) using multiple types of cells in a facile manner.

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References

  1. 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

    Article  Google Scholar 

  2. Sun W, Starly B, Daly AC et al (2020) The bioprinting roadmap. Biofabrication 12(2):022002. https://doi.org/10.1088/1758-5090/ab5158

    Article  Google Scholar 

  3. Levato R, Jungst T, Scheuring RG et al (2020) From shape to function: the next step in bioprinting. Adv Mater 32(12):1906423. https://doi.org/10.1002/adma.201906423

    Article  Google Scholar 

  4. Ng WL, Chan A, Ong YS et al (2020) Deep learning for fabrication and maturation of 3D bioprinted tissues and organs. Virtual Phys Prototyp 15(3):340–358. https://doi.org/10.1080/17452759.2020.1771741

    Article  Google Scholar 

  5. Lee JM, Ng WL, Yeong WY (2019) Resolution and shape in bioprinting: strategizing towards complex tissue and organ printing. Appl Phys Rev 6(1):011307. https://doi.org/10.1063/1.5053909

    Article  Google Scholar 

  6. He JK, Mao M, Li X et al (2021) Bioprinting of 3D functional tissue constructs. Int J Bioprinting 7(3):1–2. https://doi.org/10.18063/ijb.v7i3.395

    Article  Google Scholar 

  7. Ng WL, Wang S, Yeong WY et al (2016) Skin bioprinting: impending reality or fantasy? Trends Biotechnol 34(9):689–699. https://doi.org/10.1016/j.tibtech.2016.04.006

    Article  Google Scholar 

  8. Guo F, Li P, French JB et al (2015) Controlling cell–cell interactions using surface acoustic waves. Proc Natl Acad Sci USA 112(1):43–48. https://doi.org/10.1073/pnas.1422068112

    Article  Google Scholar 

  9. Ng WL, Yeong WY, Naing MW (2016) Microvalve bioprinting of cellular droplets with high resolution and consistency. In: Proceedings of the International Conference on Progress in Additive Manufacturing, pp 397–402

  10. Choe YE, Kim GH (2020) A PCL/cellulose coil-shaped scaffold via a modified electrohydrodynamic jetting process. Virtual Phys Prototyp 15(4):403–416. https://doi.org/10.1080/17452759.2020.1808269

    Article  Google Scholar 

  11. Kanaki Z, Chandrinou C, Orfanou IM et al (2022) Laser-induced forward transfer printing on microneedles for transdermal delivery of gemcitabine. Int J Bioprinting 8(2):554. https://doi.org/10.18063/ijb.v8i2.554

    Article  Google Scholar 

  12. Ozbolat IT, Hospodiuk M (2016) Current advances and future perspectives in extrusion-based bioprinting. Biomaterials 76:321–343. https://doi.org/10.1016/j.biomaterials.2015.10.076

    Article  Google Scholar 

  13. 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(6):e0216776. https://doi.org/10.1371/journal.pone.0216776

    Article  Google Scholar 

  14. Ng WL, Yeong WY, Naing MW (2014) Potential of bioprinted films for skin tissue engineering. In: Proceedings of the 1st International Conference on Progress in Additive Manufacturing, pp 441–446. https://doi.org/10.3850/978-981-09-0446-3_065

  15. Meng Z, He JK, Li JX et al (2020) Melt-based, solvent-free additive manufacturing of biodegradable polymeric scaffolds with designer microstructures for tailored mechanical/biological properties and clinical applications. Virtual Phys Prototyp 15(4):417–444. https://doi.org/10.1080/17452759.2020.1808937

    Article  Google Scholar 

  16. Chand R, Muhire BS, Vijayavenkataraman S et al (2022) Computational fluid dynamics assessment of the effect of bioprinting parameters in extrusion bioprinting. Int J Bioprinting 8(2):545. https://doi.org/10.18063/ijb.v8i2.545

    Article  Google Scholar 

  17. Ng WL, Lee JM, Zhou MM et al (2020) Vat polymerization-based bioprinting—process, materials, applications and regulatory challenges. Biofabrication 12(2):022001. https://doi.org/10.1088/1758-5090/ab6034

    Article  Google Scholar 

  18. Li W, Mille LS, Robledo JA et al (2020) Recent advances in formulating and processing biomaterial inks for vat polymerization-based 3D printing. Adv Healthcare Mater 9(15):2000156. https://doi.org/10.1002/adhm.202000156

    Article  Google Scholar 

  19. Nieto D, Marchal Corrales JA, de Mora AJ et al (2020) Fundamentals of light-cell–polymer interactions in photo-cross-linking based bioprinting. APL Bioeng 4(4):041502. https://doi.org/10.1063/5.0022693

    Article  Google Scholar 

  20. Takagi D, Lin W, Matsumoto T et al (2019) High-precision three-dimensional inkjet technology for live cell bioprinting. Int J Bioprinting 5(2):208. https://doi.org/10.18063/ijb.v5i2.208

    Article  Google Scholar 

  21. Xu T, Zhao WX, Zhu JM et al (2013) Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology. Biomaterials 34(1):130–139. https://doi.org/10.1016/j.biomaterials.2012.09.035

    Article  Google Scholar 

  22. Gudupati H, Dey M, Ozbolat I et al (2016) A comprehensive review on droplet-based bioprinting: past, present and future. Biomaterials 102:20–42. https://doi.org/10.1016/j.biomaterials.2016.06.012

    Article  Google Scholar 

  23. Ng WL, Zhi Qi JT, Yeong WY et al (2018) Proof-of-concept: 3D bioprinting of pigmented human skin constructs. Biofabrication 10(2):025005. https://doi.org/10.1088/1758-5090/aa9e1e

    Article  Google Scholar 

  24. Agarwala S, Lee JM, Ng WL et al (2018) A novel 3D bioprinted flexible and biocompatible hydrogel bioelectronic platform. Biosens Bioelectron 102:365–371. https://doi.org/10.1016/j.bios.2017.11.039

    Article  Google Scholar 

  25. Li X, Liu B, Pei B et al (2020) Inkjet bioprinting of biomaterials. Chem Rev 120(19):10793–10833. https://doi.org/10.1021/acs.chemrev.0c00008

    Article  Google Scholar 

  26. Ng WL, Lee JM, Yeong WY et al (2017) Microvalve-based bioprinting—process, bio-inks and applications. Biomater Sci 5(4):632–647. https://doi.org/10.1039/C6BM00861E

    Article  Google Scholar 

  27. Jentsch S, Nasehi R, Kuckelkorn C et al (2021) Multiscale 3D bioprinting by nozzle-free acoustic droplet ejection. Small Methods 5(6):2000971. https://doi.org/10.1002/smtd.202000971

    Article  Google Scholar 

  28. Yusupov V, Churbanov S, Churbanova E et al (2020) Laser-induced forward transfer hydrogel printing: a defined route for highly controlled process. Int J Bioprinting 6(3):271. https://doi.org/10.18063/ijb.v6i3.271

    Article  Google Scholar 

  29. Saunders RE, Derby B (2014) Inkjet printing biomaterials for tissue engineering: bioprinting. Int Mater Rev 59(8):430–448. https://doi.org/10.1179/1743280414Y.0000000040

    Article  Google Scholar 

  30. Suntornnond R, Ng WL, Huang X et al (2022) Improving printability of hydrogel-based bio-inks for thermal inkjet bioprinting applications via saponification and heat treatment process. J Mater Chem B 10:5989–6000. https://doi.org/10.1039/D2TB00442A

    Article  Google Scholar 

  31. Rastogi P, Kandasubramanian B (2019) Review of alginate-based hydrogel bioprinting for application in tissue engineering. Biofabrication 11(4):042001. https://doi.org/10.1088/1758-5090/ab331e

    Article  Google Scholar 

  32. Xu C, Chai WX, Huang Y et al (2012) Scaffold-free inkjet printing of three-dimensional zigzag cellular tubes. Biotechnol Bioeng 109(12):3152–3160. https://doi.org/10.1002/bit.24591

    Article  Google Scholar 

  33. Xu T, Catalin B, Michael A et al (2009) Fabrication and characterization of bio-engineered cardiac pseudo tissues. Biofabrication 1(3):035001. https://doi.org/10.1088/1758-5082/1/3/035001

    Article  Google Scholar 

  34. Noor N, Shapira A, Edri R et al (2019) 3D printing of personalized thick and perfusable cardiac patches and hearts. Adv Sci 6(11):1900344. https://doi.org/10.1002/advs.201900344

    Article  Google Scholar 

  35. Osidak EO, Kozhukhov VI, Osidak MS et al (2020) Collagen as bioink for bioprinting: a comprehensive review. Int J Bioprinting 6(3):270. https://doi.org/10.18063/ijb.v6i3.270

    Article  Google Scholar 

  36. Lee JM, Suen SKQ, Ng WL et al (2020) Bioprinting of collagen: considerations, potentials, and applications. Macromol Biosci 21(1):2000280. https://doi.org/10.1002/mabi.202000280

    Article  Google Scholar 

  37. Yang Y, Xu RZ, Wang CJ et al (2022) Recombinant human collagen-based bioinks for the 3D bioprinting of full-thickness human skin equivalent. Int J Bioprinting 8(4):611. https://doi.org/10.18063/ijb.v8i4.611

    Article  Google Scholar 

  38. Nichol JW, Koshy ST, Bae H et al (2010) Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31(21):5536–5544. https://doi.org/10.1016/j.biomaterials.2010.03.064

    Article  Google Scholar 

  39. Bertassoni LE, Cardoso JC, Manoharan V et al (2014) Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels. Biofabrication 6(2):024105. https://doi.org/10.1088/1758-5082/6/2/024105

    Article  Google Scholar 

  40. Ng WL, Yeong WY, Naing MW (2016) Development of polyelectrolyte chitosan-gelatin hydrogels for skin bioprinting. Procedia CIRP 49:105–112. https://doi.org/10.1016/j.procir.2015.09.002

    Article  Google Scholar 

  41. Ng WL, Yeong WY, Naing MW (2016) Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering. Int J Bioprinting 2(1):53–62. https://doi.org/10.18063/IJB.2016.01.009

    Article  Google Scholar 

  42. Frisman I, Seliktar D, Bianco-Peled H (2011) Nanostructuring PEG-fibrinogen hydrogels to control cellular morphogenesis. Biomaterials 32(31):7839–7846. https://doi.org/10.1016/j.biomaterials.2011.06.078

    Article  Google Scholar 

  43. Lee YB, Polio S, Lee W et al (2010) Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture. Exp Neurol 223(2):645–652. https://doi.org/10.1016/j.expneurol.2010.02.014

    Article  Google Scholar 

  44. Gao G, Yonezawa T, Hubbell K et al (2015) Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging. Biotechnol J 10(10):1568–1577. https://doi.org/10.1002/biot.201400635

    Article  Google Scholar 

  45. Gao G, Schilling AF, Yonezawa T et al (2014) Bioactive nanoparticles stimulate bone tissue formation in bioprinted three-dimensional scaffold and human mesenchymal stem cells. Biotechnol J 9(10):1304–1311. https://doi.org/10.1002/biot.201400305

    Article  Google Scholar 

  46. Gao G, Schilling AF, Hubbell K et al (2015) Improved properties of bone and cartilage tissue from 3D inkjet-bioprinted human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA. Biotechnol Lett 37(11):2349–2355. https://doi.org/10.1007/s10529-015-1921-2

    Article  Google Scholar 

  47. Jang D, Kim D, Moon J (2009) Influence of fluid physical properties on ink-jet printability. Langmuir 25(5):2629–2635. https://doi.org/10.1021/la900059m

    Article  Google Scholar 

  48. Zhang MY, Krishnamoorthy S, Song HT et al (2017) Ligament flow during drop-on-demand inkjet printing of bioink containing living cells. J Appl Phys 121(12):124904. https://doi.org/10.1063/1.4978744

    Article  Google Scholar 

  49. Xu CX, Zhang M, Huang Y et al (2014) Study of droplet formation process during drop-on-demand inkjetting of living cell-laden bioink. Langmuir 30(30):9130–9138. https://doi.org/10.1021/la501430x

    Article  Google Scholar 

  50. Blaeser A, Duarte Campos DF, Puster U et al (2016) Controlling shear stress in 3D bioprinting is a key factor to balance printing resolution and stem cell integrity. Adv Healthcare Mater 5(3):326–333. https://doi.org/10.1002/adhm.201500677

    Article  Google Scholar 

  51. Shi J, Wu B, Li SH et al (2018) Shear stress analysis and its effects on cell viability and cell proliferation in drop-on-demand bioprinting. Biomed Phys Eng Express 4(4):045028. https://doi.org/10.1088/2057-1976/aac946

    Article  Google Scholar 

  52. Luo Y, Hong YL, Shen L et al (2021) Multifunctional role of polyvinylpyrrolidone in pharmaceutical formulations. AAPS PharmSciTech 22:1–16. https://doi.org/10.1208/s12249-020-01909-4

    Article  Google Scholar 

  53. Ng WL, Yeong WY, Naing MW (2017) Polyvinylpyrrolidone-based bio-ink improves cell viability and homogeneity during drop-on-demand printing. Materials 10(2):190. https://doi.org/10.3390/ma10020190

    Article  Google Scholar 

  54. Ng WL, Ayi TC, Liu YC et al (2021) Fabrication and characterization of 3D bioprinted triple-layered human alveolar lung models. Int J Bioprinting 7(2):332. https://doi.org/10.18063/ijb.v7i2.332

    Article  Google Scholar 

  55. 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(3):562–574. https://doi.org/10.1039/C7BM01015J

    Article  Google Scholar 

  56. Liu YY, Derby B (2019) Experimental study of the parameters for stable drop-on-demand inkjet performance. Phys Fluids 31(3):032004. https://doi.org/10.1063/1.5085868

    Article  Google Scholar 

  57. Dong H, Carr WW, Morris JF (2006) An experimental study of drop-on-demand drop formation. Phys Fluids 18(7):072102. https://doi.org/10.1063/1.2217929

    Article  Google Scholar 

  58. Meyer JD, Hewlett-Packard A, Bazilevsky A (1999) Effects of polymeric additives on thermal ink jets. Recent Prog Ink Jet Technol II:450–455

    Google Scholar 

  59. Kok CM, Rudin A (1981) Relationship between the hydrodynamic radius and the radius of gyration of a polymer in solution. Die Makromol Chem Rapid Commun 2(11):655–659. https://doi.org/10.1002/marc.1981.030021102

    Article  Google Scholar 

  60. Xu DS, Sanchez-Romaguera V, Barbosa S et al (2007) Inkjet printing of polymer solutions and the role of chain entanglement. J Mater Chem 17(46):4902–4907. https://doi.org/10.1039/B710879F

    Article  Google Scholar 

  61. Zhang YZ, Hu GF, Liu YH et al (2022) Suppression and utilization of satellite droplets for inkjet printing: a review. Processes 10(5):932. https://doi.org/10.3390/pr10050932

    Article  Google Scholar 

  62. Josserand C, Thoroddsen ST (2016) Drop impact on a solid surface. Annu Rev Fluid Mech 48:365–391. https://doi.org/10.1146/annurev-fluid-122414-034401

    Article  MathSciNet  MATH  Google Scholar 

  63. Yarin AL (2006) Drop impact dynamics: splashing, spreading, receding, bouncing. Annu Rev Fluid Mech 38:159–192. https://doi.org/10.1146/annurev.fluid.38.050304.092144

    Article  MathSciNet  MATH  Google Scholar 

  64. Rioboo R, Tropea C, Marengo M (2001) Outcomes from a drop impact on solid surfaces. Atom Sprays. https://doi.org/10.1615/AtomizSpr.v11.i2.40

    Article  Google Scholar 

  65. Mundo C, Sommerfeld M, Tropea C (1995) Droplet-wall collisions: experimental studies of the deformation and breakup process. Int J Multiph Flow 21(2):151–173. https://doi.org/10.1016/0301-9322(94)00069-V

    Article  MATH  Google Scholar 

  66. Moreira ALN, Moita AS, Panão MR (2010) Advances and challenges in explaining fuel spray impingement: how much of single droplet impact research is useful? Prog Energy Combust Sci 36(5):554–580. https://doi.org/10.1016/j.pecs.2010.01.002

    Article  Google Scholar 

  67. Wal RLV, Berger GM, Mozes SD (2006) The splash/non-splash boundary upon a dry surface and thin fluid film. Exp Fluids 40(1):53–59. https://doi.org/10.1007/s00348-005-0045-1

    Article  Google Scholar 

  68. Ng WL, Huang X, Shkolnikov V et al (2022) Controlling droplet impact velocity and droplet volume: key factors to achieving high cell viability in sub-nanoliter droplet-based bioprinting. Int J Bioprinting 8(1):424. https://doi.org/10.18063/ijb.v8i1.424

    Article  Google Scholar 

  69. Takamatsu H, Rubinsky B (1999) Viability of deformed cells. Cryobiology 39(3):243–251. https://doi.org/10.1006/cryo.1999.2207

    Article  Google Scholar 

  70. Nooranidoost M, Izbassarov D, Tasoglu S et al (2019) A computational study of droplet-based bioprinting: effects of viscoelasticity. Phys Fluids 31(8):081901. https://doi.org/10.1063/1.5108824

    Article  Google Scholar 

  71. Ng WL, Yeong WY (2019) The future of skin toxicology testing-3D bioprinting meets microfluidics. Int J Bioprinting 5(2.1):237. https://doi.org/10.18063/ijb.v5i2.1.237

    Article  Google Scholar 

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Acknowledgements

This study was supported under the RIE2020 Industry Alignment Fund—Industry Collaboration Projects (IAF-ICP) Funding Initiative, as well as cash and in-kind contribution from the industry partner, HP Inc., through the HP-NTU Digital Manufacturing Corporate Lab. We would also like to acknowledge and thank the D300e HP team for supplying the cell-dispensing cassettes for the experiments. Wei Long Ng would like to acknowledge support from NTU Presidential Postdoctoral Fellowship.

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Authors and Affiliations

  1. HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University (NTU), Singapore, 637460, Singapore

    Wei Long Ng, Xi Huang, Ratima Suntornnond & Wai Yee Yeong

  2. Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University (NTU), Singapore, 639798, Singapore

    Wei Long Ng & Wai Yee Yeong

  3. HP Inc., Palo Alto, CA, 94304, USA

    Viktor Shkolnikov

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  1. Wei Long Ng
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WLN: conceptualization, methodology, investigation, visualization, writing—review & editing, and supervision. XH: investigation and writing.VS: investigation and writing. RS: investigation and visualization. WYY: funding acquisition and review.

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Ng, W.L., Huang, X., Shkolnikov, V. et al. Polyvinylpyrrolidone-based bioink: influence of bioink properties on printing performance and cell proliferation during inkjet-based bioprinting. Bio-des. Manuf. 6, 676–690 (2023). https://doi.org/10.1007/s42242-023-00245-3

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