Inkjet Bioprinting of 3D Silk Fibroin Cellular Constructs Using Sacrificial Alginate

Abstract

Silk fibroin is a natural protein which has shown great promise for tissue engineering but is not printable due to slow gelation or harsh gelation conditions which are not cell-friendly. In this study, a two-step gelation process is proposed for the printing of silk fibroin, which utilizes alginate as a sacrificial hydrogel during an inkjetting-based process. A cell-laden blend of alginate with silk fibroin is utilized to achieve rapid gelation by calcium alginate formation during printing; it is followed by horseradish peroxidase (HRP) catalyzed covalent cross-linking of the fibroin protein at tyrosine residues after printing. This two-step gelation process successfully enables 3D bioprinting of well-defined cell-laden silk fibroin constructs suitable for long-term culture. The constructs remain intact after calcium chelation to liquefy the alginate component, demonstrating the formation of silk fibroin hydrogel. NIH 3T3 fibroblasts proliferate and spread through the hydrogel after printing. Increasing metabolic activity is observed for 5 weeks after printing, and histology shows dense cell populations in cultured constructs. The proposed two-step gelation technique is expected to enable 3D silk fibroin printing for various applications.

A universal self‐eroding sacrificial bioink that enables bioprinting at room temperature

“…The sacrificial bioink developed in this study is composed of biocompatible biopolymers and circumvents all these problems. Since it is not crosslinked and erodes spontaneously, it is better than crosslinked alginate, which requires the use of citrate for its dissolution . Since citrate chelates the calcium ions, the calcium dependent cell attachment is hampered upon its use.…”

“…Therefore, approaches such as physical/chemical crosslinking, direct bioprinting in another supportive biodegradable hydrogel, bioprinting using cell‐laden hydrogel with another more viscous sacrificial hydrogel or hybrid bioprinting of biodegradable synthetic polymer shell with cell‐laden hydrogel bioinks are used to preserve the desired geometry . For a better shape fidelity, sacrificial bioinks are widely used to provide integrity to the bioprinted tissue by means of supporting the cell‐laden hydrogel …”

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications

“…Silk-gelatin bioinks have also been used to encapsulate hMSCs [345] and human mesenchymal progenitor cells [346]. Silk has been combined with alginate as a sacrificial hydrogel for inkjet printing of well-defined constructs with steady fibroblast proliferation after five weeks [347]. Table 2.…”

Reverse-engineered Silk Hydrogels for Cell and Drug Delivery

“…Chemical crosslinking has also been piloted; for example, combining silk with alginate and horseradish peroxidase allowed the development of a two-step cross-linking protocol [98]. First, the mixture was printed into a CaCl 2 bath to induce ionic cross linking of alginate; the alginate network immobilised the silk and horseradish peroxidase.…”

“…Next, addition of hydrogen peroxide catalysed the chemical cross-linking of tyrosine residues within the silk sequence. Addition of sodium citrate dismantled the ionic crosslinks of the alginate and subsequent washing removed the sacrificial alginate from the system [98].…”