# 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 results of the qualitative evaluation of cell viability and cell proliferation inside the printouts, presented in Figure 6, prove that the structures are characterized by a high survival rate of biological material embedded in the polymer matrix each time. The morphology of the cells contained inside the polymer matrix undergoes changes during cultivation--after the 3D bioprinting process, the cells are initially characterized by a spherical, spheroidal shape, which is also observed during the first week of incubation. Then, when the cultivation time of the bioprinted structures is extended to 3 weeks, the fibroblast cells achieve the ability to proliferate, grow, and spread in the hydrogel matrix. After 4 weeks of bioprinting incubation, the presence of a more homogeneous, compacted, and interconnected network of cells with spindle-shaped morphology is observed inside the hydrogel matrix. The live/dead assay results clearly indicate excellent interaction and communication between cells. After 6 weeks of cultivation, the presence of an even, interconnected, and strongly developed fibroblast cell network is observed throughout the printout volume. Macroscopic evaluation of the samples also indicates that with the increase in the degree of cellular overgrowth, a change in the color of the tubular walls of the structures from a transparent hydrogel to a milky white tissue structure is observed. Therefore, it can be concluded that fibroblast cells remodel the hydrogel matrix during ongoing cultivation. This process may occur thanks to the suitable properties of the  
Figure 5: Results of the assessment of changes in cell viability of the immortalized fibroblasts of the NIH/3T3 line contained in the hydrogel printouts during different incubation periods of the structures. hydrogel material, enabling the diffusion of nutrients and metabolites, cell adhesion and proliferation, polymer matrix degradation, and also the deposition of a new extracellular matrix [52; 53]. The viscoelasticity of the polymer matrix generates tension to enable mechanotransduction [54]. The printouts' tubular shape and thin-walled geometry ensure the availability and kinetics of diffusion of nutrients and oxygen into the biological material contained in the polymer matrix. Indeed, the literature data show that the maximum diffusion distance of nutrients and oxygen that a cell can survive is limited to 200 \(\mu\)m [55; 56]. The printed thin-walled tubular constructs, therefore, ensure even access of nutrients to the biological material contained inside the printouts, allowing the core cells to be nourished and precluding the risk of their hypoxia [57]. The spread of cells inside the printouts with progressing cultivation time results from the presence of material degradation gradients, allowing the cells to create space to grow and spread freely toward building a network structure. Gelatin, thanks to its sensitivity to ambient temperature, provides initial printout stability during the forming of the hydrogel with extrusion techniques. Degradation of gelatin-based hydrogels occurs as a result of reversible sol-gel transformation when they pass into a liquid form above the temperature of melting point (about 27-34 \({}^{\circ}\)C) and dissociate, which leads to gradual leakage from the hydrogel matrix under printout culture conditions [58; 59; 60]. Sodium alginate-based hydrogel materials incubated in physiological solutions also release Ca\({}^{2+}\) ions cross-linking the hydrogel. This reaction occurs due to exchange with Na\({}^{+}\) ions present in the cultivation environment of the bioprinted structures. The elevated temperature of the sample cultivation environment also amplifies the speed of this process. Calcium ions diffusing into the external environment of the samples, which are responsible for the binding of alginate blocks, cause gradual degradation of the polymer matrix [60; 61].