# 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 decomposition of the hydrogel based on sodium alginate and gelatin, occurring in the manner described, allows the cells to create spaces for growth and spreading. The literature data also indicate that fibroblast cells embedded in the collagen matrix release matrix metalloproteinases, such as collagenase and gelatinase, which also affect collagen degradation and other proteins [53]. The described phenomena indicate that an important aspect of designing 3D bioprinted structures, reflecting the native tissues of the body, is the creation of hydrogel constructs that enable control of the dynamic relations between cellular growth and the degradation process of the surrounding polymer matrix [52]. Figure 6: _Cont._  
Evaluation Results of the Proliferation of Fibroblast Cells of the NIH/3T3 Line Incorporated into the Pritutouts after Individual Periods of Cultivation of the Structures Using a Scanning Electron Microscope  
The scanning electron microscopy technique allows observations of the surface of hydrogel printouts at high resolution. The SEM observations provide information on the morphology, topography, and surface roughness, as well as the porosity and microstructure of the hydrogel, which allows analysis of the gel's transport properties, which directly affect the diffusion of oxygen and nutrients. In bioprinted hydrogel structures, the SEM technique also makes it possible to visualize the morphology, degree of growth, spreading, migration, and proliferation of cells contained in the material [62; 63]. The results obtained from the evaluation of changes in the morphology of the printouts after individual cultivation periods presented in Figure 7 showed that right after the 3D bioprinting process, the surface of the hydrogel printouts is smooth. The fibroblast cells are evenly distributed inside the polymer matrix structure, and they adopt a spheroidal shape, which is a result of the rigidity of the undegraded initial polymer matrix [64; 65]. During the first week of bioprinted structure cultivation, the formation of protrusions in the hydrogel structure by individual cells is observed. After three weeks of cultivation of bioprinted structures, small clusters of spreading cells are observed to appear. The SEM results confirm the data obtained using the live/dead assay, and these results indicate that after 4 weeks of incubation, a  
Figure 6: Summary of the results of assessing the viability and proliferation of cells contained in hydrogel printouts obtained using the live/dead assay. Arrows indicate the presence of spheroidal agglomerates. Elaborated by the authors using BioRender.com [46]. significant aggregation of biological material takes place in the bioprinted structure towards the formation of an evenly distributed cellular network in the polymer matrix in place of single spheroidal agglomerates. As the cultivation time of the bioprinted structures increases, increasingly dense, clearly spreading network structures of fibroblast cells with an elongated, spindle-shaped morphology are observed. After 6 weeks of cultivation, they form a homogeneous tissue structure over the entire volume of the hydrogel. The results of observation of bioprinted structures after 8 weeks of cultivation indicate the presence of a growing amount of biological material in the produced cell network but a change in cell morphology from an elongated shape to densely arranged cells next to each other with a renewed spherical morphology. During the cultivation of the bioprinted structures, the surface morphology of the samples also changes, which, from being initially smooth, adopts a more developed and wavy structure. This observation may be due to the degradation of the polymer matrix occurring due to the release of gelatin from it because, with the decrease in the gelatin content in the polymer matrix, the size of the pores increases. In turn, the porous structure provides living space for cells; therefore, as the porosity increases, facilitated proliferation and spreading of cells in the polymer matrix occurs [62; 66].