3D printed hydrogels with oxidized cellulose nanofibers and silk fibroin for the proliferation of lung epithelial stem cells

A novel biomaterial ink consisting of regenerated silk fibroin (SF) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized bacterial cellulose (OBC) nanofibrils was developed for 3D printing lung tissue scaffold. Silk fibroin backbones were cross-linked using horseradish peroxide/H2O2 to form printed hydrogel scaffolds. OBC with a concentration of 7wt% increased the viscosity of inks during the printing process and further improved the shape fidelity of the scaffolds. Rheological measurements and image analyses were performed to evaluate inks printability and print shape fidelity. Three-dimensional construct with ten layers could be printed with ink of 1SF-2OBC (SF/OBC = 1/2, w/w). The composite hydrogel of 1SF-1OBC (SF/OBC = 1/1, w/w) printed at 25 °C exhibited a significantly improved compressive strength of 267 ± 13 kPa and a compressive stiffness of 325 ± 14 kPa at 30% strain, respectively. The optimized printing parameters for 1SF-1OBC were 0.3 bar of printing pressure, 45 mm/s of printing speed and 410 μm of nozzle diameter. Furthermore, OBC nanofibrils could be induced to align along the print lines over 60% degree of orientation, which were analyzed by SEM and X-ray diffraction. The orientation of OBC nanofibrils along print lines provided physical cues for guiding the orientation of lung epithelial stem cells, which maintained the ability to proliferate and kept epithelial phenotype after 7 days’ culture. The 3D printed SF-OBC scaffolds are promising for applications in lung tissue engineering.