Bioprinting multidimensional constructs: a quantitative approach to understanding printed cell density and redistribution phenomena

Currently, bioprinting high cellular fractions in bio-inks represents a critical step towards engineered tissue models with enhanced biological relevance. However, precise spatiotemporal mapping of high cell densities within hydrogel materials represents a significant challenge. To address these needs and challenges, this paper advances a systematic multidimensional analytical framework to quantitatively determine the spatial variations in cell density and temporal evolution of cell redistribution phenomena within bioprinted cell-laden fiber-based constructs as a function of the complex interplay of the various processing parameters for an alginate-gelatin hydrogel material system. For a one dimensional analysis, fibers printed at high relative viscosities exhibit large contact angles in the cross-sectional plane with a high degree of cell dispersion (D-d = 96.8 +/- 6.27%). Conversely, fibers printed at low relative viscosities exhibit small contact angles with cells coalescing towards the fiber midline (D-d = 76.3 +/- 4.58%). The observed in-process redistribution of cells is attributed to the flow topology inside the needle tip. The precise mechanism is governed by the temperature-dependent viscoplastic material properties as demonstrated numerically using the Herschel-Bulkley model. In the 2D analysis, the printed grid structures yield differences in local cell densities between the strut and cross regions within the fibers. At low relative viscosities, cells aggregate in regions where two overlapping fibers fuse together, yielding a high cell density ratio (1.79 +/- 0.04) between the cross and strut regions. However, at high relative viscosities, the cell density ratio decreases (0.96 +/- 0.03). In the 3D analysis, due to gravity and extrusion process effects, initially uniform bio-ink cell distributions within bio-inks are redistributed as a function of printing time, yielding quantitative increases in cell density for the initial layers, followed by quantitative decreases in the subsequent layers. Finally, a time course incubation study of the 3D bioprinted cell-laden construct reveals a time-dependent increase in cell density owing to proliferation that accelerates the material degradation.