# Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth
## 3 Results and Discussion  
### Results of the Evaluation of the Rheological Properties of the Polymer Solutions  
The evaluation of the rheological properties of polymer solutions is a key parameter to determine flow properties and shape fidelity, which affect the printability of biofluk. The rheological characteristics of the hydrogel composition also directly influence the rate of proliferation and differentiation of cells contained in the polymer matrix [47]. The results of the analysis of the rheological properties of polymer solutions based on sodium alginate and gelatin, shown in Figure 4, indicate that both compositions--both 2A9G supplemented with biological material and not containing cells--have the features of non-Newtonian systems and are characterized by shear-thinning ability. This property is characteristic of sodium alginate-based hydrogel solutions [48; 49; 50]. It enables a process of smooth printability and increased printout accuracy after the manufacturing process [47; 50]. The evaluation of the rheological properties of the two polymer solutions also indicates that the content of biological material in the biofluk in the form of immortalized cells of fibroblast NIH/3T3 line with a density of 1\(\cdot\)10\({}^{6}\)/mL causes a decrease in viscosity with respect to the composition containing no biological material. Research work carried out by the team of [PERSON] et al. confirms that as the density of the biological material in the sodium alginate and gelatin-based hydrogel bioink increases, a gradual decrease in the viscosity of the polymer solution occurs. Indeed, the cell content of the biofluk can affect the gel microstructure by blocking inter-chain interactions in the polymer solution [47; 48]. Higher viscosity hydrogel compositions require higher pressure to be applied during extrusion, which results in higher shear stresses on the cells contained in the biofluk, leading to cell damage during the fabrication of bioprinted structures [49; 51]. Therefore, the observed viscosity decrease in the polymer composition supplemented with biological material is an advantageous factor for obtaining high cell viability in the fabricated bioprinted hydrogel structures. Results of the Assessment of the Viability of Fibroblast Cells of the NIH/3T3 Line Contained in Hydrogel Printouts after Individual Periods of Cultivation of Structures Using the Fluorescent Live/Dead Assay  
Presented in Figure 5, the results of the cell viability assessment of the immortalized fibroblasts of the NIH/3T3 line contained in the hydrogel printouts indicate that directly after the 3D bioprinting process, the average survival rate of the biological material is equal to 88.91 \(\pm\) 7.50%. This observation makes it possible to state that the very process of manufacturing the tubular structures using the 3D extrusion bioprinting technique does  
Figure 4: Results of the evaluation of the rheological properties of polymer solutions containing no biological material and hydrogel compositions supplemented with fibroblast cells of the NIH/3T3 line. not have a destructive effect on the viability of model line cells incorporated into the bioink. The high viability of the cells in the printouts proves the excellent biocompatibility of the polymer matrix based on sodium alginate and gelatin. After 7-day cultivation, the viability of fibroblast cells contained in hydrogel printouts increases, reaching an average value equal to 98.73 \(\pm\) 1.32%. After 3 weeks of cultivation, the viability of fibroblasts inside the bioprinted structures reaches an average value of 97.74 \(\pm\) 1.84%. Observations of the degree of proliferation of the biological material contained inside the printouts after 3 weeks of cultivation indicate that the cells begin to spread in the hydrogel matrix and agglomerate toward each other, forming larger clusters. The increased number of dense clusters of biological material inside the printouts during later cultivation periods makes it impossible to clearly quantify cell viability using the live/dead assay.