Poster M19 - Specific Salt Ion Effects on 3D-Printed Silk Fibroin Tissue Scaffolds

Introduction: 3D-printed silk fibroin structures are promising scaffolds for tissue regeneration and drug delivery, primarily due to manufacturing flexibility, proteinaceous composition, and minimal immunogenicity. However, the properties of these silk scaffolds are usually challenging to be finely manipulated or will be compromised by harsh processing conditions. Here, we propose a bioinspired approach that exploits salt ions to tune the molecular assembly pathway of silk proteins and thus, the morphological and other properties at the macroscopic level. Salt ions play a central role in the silk spinning of both silkworms and spiders by constituting the solvent environment of the native spinning glands. The capability to tune silk fibroin scaffolds using salt ions under ambient and aqueous conditions holds great potential in hoaxing cell differentiation and facilitating tissue morphogenesis in vitro.

Materials and

Methods: Trapezoidal solid silk films were printed by extruding a monolithic silk fibroin ink to a 4.4 M sodium chloride bath, as described in our previous work. The impact of potassium, sodium, ammonium, chloride, hydrogen phosphate, and sulfate ions on 3D-printed silk fibroin films was studied by a 24-hour treatment in various salt baths consisted of these ions, followed by an additional 24-hour treatment in a 2.3 M ammonium sulfate solution to stabilize the proteinaceous structures. We used a microplate reader, Scanning Electron Microscopy (SEM), Raman spectroscopy and uniaxial tensile test to characterize the transparency, morphology, molecular conformation and mechanical properties of the 3D-printed silk fibroin films, respectively. Human primary bronchial epithelial cells were cultured on the treated films to evaluate their cytocompatibility using confocal microscopy and cell proliferation assay.

Results, Conclusions, and Discussions: The properties of 3D-printed silk films are manipulated via tuning salt composition and concentration. In particular, the magnitude of transparency is almost proportional to the concentration of K2HPO4. We also found the effect of salt ions on the morphology, such as porosity, characterized by SEM, molecular conformation characterized by Raman, and mechanical properties characterized by uniaxial tensile test. Potassium and sodium are distinct in the capability to induce the formation of beta-sheets, resulting in distinct mechanical properties. Specifically, NaCl-treated film showed a higher ductility whereas KCl-treated film had a higher Young’s modulus. More importantly, we found there is a clear correlation between the rank of salt ions in the Hofmeister series, transparency, morphological features, and mechanical properties, thus implying a salt ion-based mechanism for manipulating silk fibroin scaffolds. The cytocompatibility of silk films under salt treatments is confirmed by the formation of monolayer of human primary epithelial bronchial cells and cell proliferation assay.
Our study demonstrates the effect of salt ions, inspired by silk spinning, on the properties of 3D-printed silk films. Most likely governed by the Hoffmeister series, salt ion composition dictates silk protein assembly pathways, affecting surface and cross-sectional morphology and thus, macroscopic properties. These findings highlight the potential of monolithic proteinaceous compositions and ambient, aqueous 3D-printing for various biomedical applications such as bioengineered airway patches.

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