Fig. 4.

Cell seeding density influence on 3D cell-laden biomaterial strands and nozzle extrusion.

(a) Extruded cell-laden filaments can retain a number of cells proportional to the cell seeding density. Polymeric chains (dark blue) concentration and distribution directly influence cell proliferation capability. A lower cell seeding density results in poor cell distribution within the printed strand showing low cell-to-cell interaction and a limited proliferation rate. Increasing the cell seeding density results in a printed strand filled with cells with a high degree of cell-to-cell interaction and cell death. Proliferation and viability is limited by physical stresses imposed by neighbouring cells. Cell density can be tuned to achieve an even distribution of cell encapsulated within the printed filament in such a way that cells can maintain the required interaction with other cells to remain mitotically active and proliferate. (b) Maintaining constant the number of cells loaded in a printing syringe but changing the nozzle aperture will affect cell printability. Large nozzles (>800 μm) allow limited cell-nozzle walls and cell-to-cell interactions resulting in a widespread distribution of cells in suspension within the bioink. These settings can ensure high cell survival upon extrusion but result in low resolution of the overall construct. In contrast, narrower nozzles (<250 μm) offering smaller surface area for the same number of cells force biopaste encapsulated cells to interact with one another resulting in high density at the nozzle aperture. A narrow orifice can produce high resolution, as well as high cell death upon printing. Medium size conical nozzles (250 μm-800μm) ensure an optimal cells distribution within the nozzle and an increase in print resolution without influencing, extensively, cell survival.