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3D bioprinted silk fibroin hydrogels for tissue engineering

Nature Protocols volume 16, pages 5484–5532 (2021)Cite this article

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Abstract

The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2–9 weeks.

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Fig. 1: Overview of the procedure for fabrication of Sil-MA bioink and Sil-MA hydrogel using a DLP 3D printer.
Fig. 2: Photographs of the process for preparation of Sil-MA bioink and hydrogel.
Fig. 3: Printability of Sil-MA bioink and its application for cartilage tissue engineering.
Fig. 4: Process flow for trachea tissue engineering using DLP 3D printed cell-laden Sil-MA hydrogel36.
Fig. 5: Trachea tissue engineering using DLP 4D bioprinted cell-laden Sil-MA hydrogel70.
Fig. 6: Application of Sil-MA bioink as an electroconductive bioink for neurogenesis71.
Fig. 7: Characterization of Sil-MA solution and Sil-MA hydrogel fabricated by DLP 3D printer17.
Fig. 8: Physical, morphological and biological characterization of Sil-MA hydrogel fabricated using DLP 3D printer17.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers: ref. 17 (https://doi.org/10.1038/s41467-018-03759-y), ref. 36(https://doi.org/10.1016/j.biomaterials.2019.119679), ref. 70 (https://doi.org/10.1016/j.biomaterials.2020.120281), ref. 71 (https://doi.org/10.1021/acs.nanolett.0c02986), and ref. 68 (https://doi.org/10.1038/s41427-020-0227-6).

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Acknowledgements

This work was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (HI20C0408), the Industrial Technology Alchemist Project (20012327) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea), and Hallym University Research Fund.

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  1. These authors contributed equally: Soon Hee Kim, Heesun Hong, Olatunji Ajiteru, Md. Tipu Sultan.

Authors and Affiliations

  1. Nano-Bio Regenerative Medical Institute, College of Medicine, Hallym University, Chuncheon, Republic of Korea

    Soon Hee Kim, Heesun Hong, Olatunji Ajiteru, Md. Tipu Sultan, Young Jin Lee, Ji Seung Lee, Ok Joo Lee, Hanna Lee, Hae Sang Park, Kyu Young Choi, Joong Seob Lee & Chan Hum Park

  2. Departments of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, School of Medicine, Hallym University, Chuncheon, Republic of Korea

    Hae Sang Park & Chan Hum Park

  3. Department of Otorhinolaryngology, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea

    Kyu Young Choi

  4. Department of Otorhinolaryngology, Hallym University Sacred Heart Hospital, Anyang, Republic of Korea

    Joong Seob Lee

  5. Nano-Bio Regenerative Technology Company Ltd., Chuncheon, Republic of Korea

    Hyung Woo Ju

  6. Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, Republic of Korea

    In-Sun Hong

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  1. Soon Hee Kim
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Contributions

All authors contributed to experimental work shown in this protocol. C.H.P. supervised the project and provided funding. S.H.K. conceived and managed the manuscript preparation. S.H.K., H.H., O.A. and M.T.S. drafted and revised the manuscript with input from all authors. Y.J.L., J.S.L., O.J.L., H.L., H.S.P., K.Y.C., J.S.L., H.W.J. and I.S.H. partially wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Chan Hum Park.

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Key references using this protocol

Kim, S. H. et al. Nat. Commun. 9, 1–14 (2018): https://doi.org/10.1038/s41467-018-03759-y

Hong, H. et al. Biomaterials 232, 119679 (2020): https://doi.org/10.1016/j.biomaterials.2019.119679

Kim, S. H. et al. Biomaterials 260, 120281 (2020): https://doi.org/10.1016/j.biomaterials.2020.120281

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Supplementary Data 1

Square model for cytocompatibility test

Supplementary Data 2

Human trachea CAD model

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Kim, S.H., Hong, H., Ajiteru, O. et al. 3D bioprinted silk fibroin hydrogels for tissue engineering. Nat Protoc 16, 5484–5532 (2021). https://doi.org/10.1038/s41596-021-00622-1

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