Stabilizing Gelatin-Based Bioinks under Physiological Conditions by Incorporation of Ethylene-Glycol-Conjugated Fmoc-Ff Peptides

Peptide-based bioinks that stabilize gelatin-based 3D printed scaffolds under physiological conditions. 

Gelatin is one of the most widely-used materials for the fabrication of 3D scaffolds. In order to fabricate stable scaffolds under physiological conditions, gelatin need to be methacrylated, allowing the crosslinking and therefore the stabilization of the hydrogel through UV treatment. However, the UV and the crosslinking agents are often toxic for cell viability. 

We have developed new bioink formulations for 3D-printing by co-assembly of gelatin with the low molecular weight peptide gelator fluorenylmethoxycarbonyl diphenylalanine (FmocFF) and its ethylene glycol conjugate (Fig.1a). The new bioinks do not require the use of any crosslinking agent, harnessing the ability of the short peptides to self-assemble to obtain a stable hydrogel scaffold (Fig. 1b). Changing the ratio between the peptides building blocks allowed to tune the mechanical properties of the hydrogels (Fig. 1c). The combination of the peptides and the gelatin enhances the printability of the hydrogel. The conjugation of ethylene glycol contributes to the precision of the 3D-printing of the hybrid hydrogels, and the fabrication of 3D-printed constructs. Furthermore, the 3D-printed constructs are highly biocompatible, and support cell viability (Fig. 1d).

Figure 1: (a) Schematic representation of the bioionk preparation. (b) Optical images of the 3D-printed composite hydrogels constructs. (c) Storage modulus (G′) of the composite hydrogels at 25 and 37 °C. (d) Cell growth supported by the 3D-printed ethylene glycol conjugated Fmoc-FF/Gel constructs, demonstrating the biocompatibility of the scaffolds.

1. Bioink for 3D printing of scaffolds for cell growth and for tissue engineering
2. Scaffold for developing 3D organoid cultures
3. Delivery of bioactive molecules, growth factors and drugs which can be incorporated into the hydrogel and released in a sustained manner.

As verified by in vitro assay results, the scaffolds’ topography and surface chemistry promote mesenchymal stem cells (MSCs) and fibroblast proliferation. By incorporating the ethylene glycol motif into the peptide design, we enhance the printability properties of the new bioink formulation. Finally, the multi-component hydrogels can be functionalized by immobilization of biologically active molecules (such as the RGD peptide) on the surface, or by the incorporation of growth factors, thereby improving MSCs viability and enhancing the regenerative properties of the scaffolds for different specific tissue regeneration applications.

US Provisional Patent .

Stabilizing gelatin-based bioinks under physiological conditions by incorporation of ethylene-glycol-conjugated Fmoc-FF peptides. F. Netti, M. Aviv, Y. Dan, S. Rudnick-Glick, M. Halperin-Sternfeld, L. Adler-Abramovich. Nanoscale; 2022 14, 8525-8533

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