4.6.1 Correlation of shear stress and cell vitality
To solve the question of whether it is reasonable to improve printing resolution by increasing the shear stress, evaluating the impact of shear stress on living cells is essential. For this purpose, we investigated the short-term and possible long-term effects of printing-induced shear stress on primary porcine hip arterial endothelial cells (pHAE). We printed pHAE from five independent donors (n = 5) at three different shear stress levels (20, 30, and 40 kPa) and recorded the resultant cell viability (Figure 10a–j). With increasing shear stress, as shown in Figure 12, the average cell viability in the kc composite was significantly decreased from 56.2% (20 kPa) to 42.1% (40 kPa). Nonetheless, the cell viability in kc-s and kc-c seemed unaffected (with >93% average cell viability). Moreover, the viability of the cells in kc-sa-c increased with the printing pressure increasing from 94.7% (20 kPa) to 95.8% (30 kPa). Our results indicated that a higher shear stress may stimulate cell proliferation, as has been reported[42-45]. To understand the effect of shear stress on quantificational cell viability, the distribution of dead cells was assayed using the equal area method. The cross-section of the gel filament was divided into three concentric annulus areas (Figures 12a,1, 2, 3) from the core to the shell. Meanwhile, several dead cells in each area were calculated and averaged. As shown in Figure 12k–m), from the centre (area 1) to the outer (area 3) area, the average cell viability in kc significantly decreased from 60.7% (20 kPa) to 56.3% (20 kPa).A similar conclusion was reached at 30 and 40 kPa (Figure 12k). The average cell viability in kc-sa (Figure 10l) and kc-c (Figure 10m) showed a similar gradient, and the difference in each area was approximately 5%.Due to the gradient distribution of shear stress the cells in the centre layer of the gel filament received the least shearing force, such that the number of dead cells was the least. Secondly, the cells in the middle layer received moderate shearing force. The cells in the outermost layer received the largest shearing force, showing the maximum number of dead cells. The growth of cells exposed to different shear stress levels differed with the printing process. However, to elucidate this phenomenon, further mechanobiological studies with a focus on the intracellular mechanisms in response to shear stress should be conducted.