# 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 research conducted by the team of [PERSON] et al. indicates that human adult dermal fibroblasts of NHDF-Ad need about 14 days to begin the process of spreading in a plasma-agarose hydrogel matrix prepared based on 1% agarose. Cell spreading and cell migration are hindered in materials with small and rigid pores, preventing the movement of cell nuclei [67]. Degradation of the polymer matrix also changes its viscoelastic properties, and the processes that occur in the matrix architecture affect both its viscosity and susceptibility to degradation [68]. The spreading and migration of cells in the structure of bioprints are also promoted by the increased plasticity and limited rigidity of the polymer matrix due to degradation [64; 65]. This is because it has been shown that the relationship between gel rigidity and cell spreading ability is contradictory between 2D and 3D culture conditions [65]. The cells contained inside the hydrogel structures are capable of migrating in a protease-independent manner when the polymer matrices exhibit sufficient mechanical plasticity [64; 68]. The work of the team of [PERSON] et al. indicates that the cells contained in a three-dimensional matrix initially exert deformations, which induce stresses determined by the initial elastic modulus of the matrix. In viscoelastic matrices, these forces can undergo relaxation due to mechanical deformation and remodeling of the polymer matrix. The rate of stress relaxation determines the degree of mechanical remodeling of the matrix. In rapidly relaxing matrices, this facilitates the clustering of adhesion ligands, cell shape change, spreading, and proliferation [69]. The preparation process of bioprinted hydrogel structures necessary for SEM observations based on dehydrating the samples initially and drying at the critical point may result in the shrinkage and thickening of the material structure. For this reason, there is a risk that the observed morphology of the samples does not reflect the original state of the hydrogel, which undergoes changes in the swollen condition [67; 70]. For this reason, SEM microscopic observations can provide a comparative verification technique to other techniques for visualizing bioprinted hydrogel structures. Nevertheless, a key aspect of the SEM microscopic observations of the bioprinted structures was to assess the distribution, morphology, and proliferation status of the biological material in the printouts. These parameters are not directly dependent on the hydration and preparation method of the structures and prove the complete cellular hypertrophy of the printouts, which is homogeneous in the volume of the material, during their long-term cultivation conducted within this study's framework. Figure 7: Summary of the analysis of changes in cell morphology contained in hydrogel printouts using SEM microscopic observations. Arrows indicate the presence of spindle-shaped fibroblasts. Elaborated by authors using BioRender.com [46]. Evaluation Results of the Mechanical Properties of Bioprinted Hydrogel Structures Subjected to Individual Cultivation Periods Using a Static Tensile Test  
The results of the evaluation of the tensile strength of bioprinted hydrogel structures, shown in Figure 8, indicate that the printouts are characterized by very poor mechanical properties directly after the fabrication process of tubular structures. The average maximum printout breaking force equal to 0.19 \(\pm\) 0.07 N is characterized by samples containing biological material in their structure. A slightly lower tensile strength of about 0.14 \(\pm\) 0.12 N was registered for printouts that did not contain fibroblast cells. Thus, we can see that the presence of biological material in the polymer matrix allows for improving the mechanical properties of hydrogel printouts. For hydrogel structures, the rigidity of the material plays a key role in having a direct impact on the cellular activity and remodeling of the polymer matrix.