Page 109 - IJB-8-4

Köpf, et al.
behavior of shear thinning fluids were calculated. For later on can be used for proper tuning of the bioprinting
a 1.5% alginate solution with a cell concentration of 1 process.
million cells/ml, we found n to be 0.944 ± 0.004 and K to The following cell culture results are presented in
be 0.193 ± 0.013 Pa·s . To validate the applied simulation combination with the applied printing pressure ranging
n
model, the calculated droplet volumes (from simulation) from 0.25 to 3.0 bar. This printing or upstream pressure
were compared with experimentally measured ones for can be precisely set before printing and is a controlling
two upstream pressures of 0.5 and 3 bar (Figure 1A). parameter in the process. According to the results
The simulation predicts slightly lower droplet sizes: presented in Figure 1, high pressure values are associated
The deviation in droplet volumes calculated from the with higher levels of the corresponding average and
simulation compared to the measured ones was 19.5% and maximum shear stress and thus a higher loss in cell
18.9% for 0.5 and 3 bar, respectively. The corresponding viability. After being expelled from the printing nozzle,
average wall shear stress occurring inside the nozzle was the viability of HUVECs decreased with increasing
1.62 and 9.19 kPa for the upstream pressures 0.5 and printing pressure (Figure 2). The normalized viability
3.0 bar. values decreased almost steadily and ranged from 100%
Figure 1B shows the maximum and average wall for 0.25 bar to 79.7% for 3.0 bar.
shear stress within the critical region of the microvalve In the non-printed as well as in all printed samples, 2D
nozzle. Here, the maximum wall shear stress is simply network-like structures of similar quality and comprising
the maximum over the wall of the microvalve nozzle viable endothelial cells could be observed (Figure 3A).
(automatically calculated by software) and the average Previously, printed cells were exposed to different
wall shear stress is the area average over the whole shear stress levels corresponding to the applied printing
microvalve nozzle wall. The average increases in an pressures of 0.5 and 3.0 bar. Software-based image analysis
almost linear fashion with the increasing pressure. While revealed no statistically significant differences between
the average wall shear stress is always below 10 kPa, the the number of crossing points of cellular structures, the
maximum reaches the value of 21 kPa at 3 bar. However, number of closed network elements, and the number of
not all the cells traveling through the microvalve would extensions or dead ends (P < 0.05, Figure 3B). However,
experience such high values of shear stress occurring in samples printed under the harshest printing conditions
at or near the nozzle walls. To demonstrate this, we (3 bar), significantly more dead cells were found than in
considered three streamlines passing from 15 to 30, and all other samples (P < 0.05, Figure 3C).
45 µm radial distances away from the centerline of the We further demonstrated that the applied printing
nozzle (Figure 1C). Assuming cells as mass-free particles conditions did not alter the expression of endothelial
travelling within the microvalve on the flow streamlines, cell specific markers such as vascular-endothelial (VE)
they experience the fluid shear stress and pressure cadherin and von Willebrand factor (Figure 4). As
occurring in each spatial position along the streamline. confirmed by visual examination, both markers were
Figure 1D and E show the pressure and shear stress equally present in non-printed controls and in samples
along the three streamlines. The figures show that almost printed at 0.5 and 3.0 bar. The cell nuclei were visualized
all the cells traveling through the microvalve experience by DAPI staining. In all samples, VE cadherin was highly
the same hydrostatic pressure regardless of their radial concentrated at the cell-cell interface. The intensity of
position. The shear stress imposed on the cells depends the signal for both target proteins was different from cell
on their radial distance from centerline; the closer to the to cell. However, there were no qualitative differences
centerline, the lower the shear stress. visible among the images from the three groups.
This study found that there are two aspects that Under 3D culture conditions and in co-culture with
should be considered when studying the effects of hMSC, endothelial cells exposed to low hydrostatic
printing processes on cells. First, since the nozzle is very pressure/shear stress formed capillary-like structures
short, the cells experience a very short time of exposure to (Figure 5). Using an established angiogenesis model
shear stress while passing through together with a jump in based on a hydrogel blend of agarose and collagen ,
[6]
shear stress (known as extensional stress) at the entrance we observed in the present work that the formation
region of the nozzle (inset, Figure 1E). Second, cells are of capillary-like structures was drastically reduced
exposed to hydrostatic pressure throughout nearly the after cells were exposed to high bioprinting-associated
entire printing time frame (because the printer reservoir hydrostatic pressure and resulting shear stress. TPLSM
is under pressure throughout the printing process) as images revealed comparable structure formation in non-
well as a high-pressure gradient during the time they printed samples and in those which were printed at 0.5
are flowing through the nozzle of the microvalve. The bar. However, in samples printed at 3.0 bar no structure
simulation results offer insights about the type, duration formation was observed at all. Here, all cells remained
and amplitude of the mechanical stimuli on the cells that rounded throughout the cultivation period.

International Journal of Bioprinting (2022)–Volume 8, Issue 4 101