## 4 Discussion  
As a current state-of-art bioprinting technique, microextrusion-based printing liquid hydrogel precursors within a supporting bath medium enable the formation of complex 3D structures. Although the essential role of a support bath in bioprinting is well-recognized, underlying structural phenomena and mechanisms are still largely unknown. In the context of emergent phenomena distinct from traditional bioprinting in air medium, control of the structural outcomes with quality design attributes requires optimization of key printing process parameters. By optimizing the bioprinting parameters  
Figure 9: The design and print of a multi-tubular hydrogel structure: (**a**) the 3D CAD model of the bifurcation structure; (**b**) customized toolpath; (**c**) live/dead cell fluorescent image of single filament with cell-laden bio-ink; (**d**) the printed structure without using optimal parameters, yielding irregular surface; (**e**) the printed structure using optimal parameters, yielding a smoother surface and enhanced shape fidelity; (**f**) Nanoclay separates the petri dish to 2 parts without water leakage; (**g**) Tubular structure is buried within nanoclay; (**h**) Water stained in red flow automatically to the left side. for filament and tubular structures within a nanoclay support bath, the optimal printing parameters are identified and reported. First, the design and implementation of a temperature control system to the bioprinter enables the support bath temperature to be stably maintained. This temperature control system is controlled by PID controller and operated with customized software with GUI. Compared to other studies that implement nanoclay as a support bath material, the findings from this paper show that the support bath temperature has a significant effect on the formation of the printed hydrogel structures. Upon lowering the support bath temperature below room temperature (22 \({}^{\circ}\)C), the issue of clogging arises, which has not been reported in previous studies [14]. Furthermore, by analyzing the single filament dimension, a relationship between the filament cross-sectional long axis with extrusion flowrate and travel speed is revealed. Based on this, the optimal layer height is determined to enable solid material fusion between successively printed layers. At first, the optimal layer height for printing tubular structure is identified as 0.5 mm when the flowrate is 4.8 mL/h and travel speed is 200 mm/min. The temperature of the support bath is set near room temperature (22 \({}^{\circ}\)C). However, to improve printing time efficiency, different travel speeds are tested, resulting in the identification of optimal parameters for printing tubular structures with uniform surface characteristics at shorter print times. Although other state-of-art studies do consider the rheological properties as major parameters, the interplay with key process parameters has not been systematically studied, along with the effect on printed 3D structure. However, this paper highlights the key process parameters that are critical for bioprinting complex structural outcomes within a support bath medium. To characterize the printed structure, the irregularity is proposed to measure the surface quality of the tubular structure. The irregularity serves as a quantitative metric that reveals the relationship between the investigated parameters and printed outcomes. Another property that is critical to the bioprinting structure is the resolution. It is reported herein that the single printed filament is on a millimeter scale, which is a relatively low resolution for micro-EBB technique. However, the print resolution can be significant improved by using smaller diameter nozzles. The resolution of the demonstrated bioprinted 3D tubular structures is still within the range of current state-of-art biopprinted structures. The typical printed tube has a 5-10 mm diameter when using pluronic F127 and GelMA blend [26].