## 4 Discussion  
In order to create complex tissue/organ structure using 3D bioprinting technology, the printability of hydrogel is a crucial aspect to allow for a suitable environment for organ functionalizations [19; 29]. When applying specific materials for bioink in micro-extrusion printing, an appropriate printing parameter should be considered to achieve optimum printing structures [28]. In this study, we presented a preliminary ideal index of 3 BE hydrogel as bioink to generate complex 3D architectures by means of self-fabricated 3D bioprinting system. We found that different combinations of printing parameters, especially the needle size and bioink concentration influence the printed results. The biomaterial properties and biological requirements of bioink may affect their performances in 3D bioprinting applications [10]. In the present study, we observed the printing quality of line width after the printing process using 3 BE-1 hydrogel under fixed needle sizes, yet we found that the 3D construct cannot be printed in all combinations made. This may be due to the low concentration of 3 BE hydrogel being used. A previous study found that the 3D bioprinting accuracy of hydrogels depends largely on polymer concentrations [30]. Low viscosities of bioink often failed to generate the 3D printing of layers [31]. Fortunately, the printability and properties of the resulting 3D constructs can  
Figure 7: Cell proliferation of L929 fibroblasts in the 3 BE-3 (\(\Phi\) 0.13 mm needle head) at days one and three after bioprinting. More cell proliferation was seen in day three compared to day one. be significantly improved by changing the concentration of the components [27]. In this study, we discovered a slight increase in the printing quality of line width when 3 BE-2 or 3 BE-3 was used compared to 3 BE-1. In addition, the image taken after printing the line using the 3 BE-2 and 3 BE-3 hydrogels indicated that both concentrations could be the ideal option to generate a precise and stable line width. Regarding the 3D construct from the printing parameter, we observed that the intended shape can be printed using the combinations of 3 BE-2. However, the printed constructs with the 3 BE-2 combinations failed after 4 h post-printing. This feature implied that the viscosity in this range (~1500 Pa-s) cannot be applied to support the 3D construct. Whereas, in order to bioprint a stable soft tissue structure, the combinations of 3 BE-3 hydrogel might be the ideal options. We found that when using these combinations, a stable and smooth 3D construct can be generated. The F127 content in the composite highly influences the printability of the hydrogel. The higher the concentration of F127 hydrogel, the better printability can be reached [19]. Altering appropriate concentration ratios of gelatin and hydrogel could generate regular and smooth fibers steadily resulting in excellent printability [32]. Furthermore, we also compiled the preliminary experiment to study the viability of L929 fibroblasts combined with 3 BE hydrogel within the printing process. It was found that more cell death was present 24 h post-printing. The decrease of cell viability after printing is the commonly encountered issues in 3D bioprinting application [33]. This might be caused by the pressure used to expel the bioink in microextrusion bioprinting [34]. Printing at higher extrusion pressures will increase the number of dead cells since a higher pressure will elevate the shear stress in the nozzle, which eventually damages the cell membrane and leads to lower cell viability after extrusion [21; 35]. A previously published study demonstrated an inverse relationship between extrusion pressure and cellular viability, especially in high extrusion pressures [34]. In addition, hydrogel concentrations can be another issue to affect cell viability after printing. One study found that a higher concentration of hydrogel showed the fastest decrease in metabolic activity of the cell compared to the low concentration group in long term observation [36].