# 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
Mechanical interactions between cells and the extracellular matrix are considered symbiotic, as there is a mutual dynamic relationship between the hydrogel, which regulates intracellular processes, and the biological material, which enables its remodeling. The viscoelastic properties of bioprinted hydrogel structures with cultivation time may vary due to a combination of shrinkage, degradation, and extracellular matrix accumulation, which can affect the water content of the material, as well as the kinetics of oxygen and nutrient diffusion [53]. This thesis is confirmed by the results obtained within the framework of this study, which indicate the occurrence of dynamic changes in the mechanical properties of bioprinted structures during the different periods of their culture. During the initial periods of cultivation of the bioprinted structures, a decrease in the tensile strength of tubular structures is observed with respect to samples not subjected to incubation. The registered average values of maximum printout breaking force are, in fact, 0.07 \(\pm\) 0.02 N, 0.12 \(\pm\) 0.07 N, and 0.10 \(\pm\) 0.04 N for cultivation times equal to 1, 3, and 4 weeks, respectively. The observed relationship may be related to the gradual degradation of the polymer matrix due to the release of gelatin from it. The results of SEM observations indicate that during the 4-week cultivation, the process of significant growth and spreading of fibroblast cells contained in tubular printouts starts to occur. However, their number during this period is not yet sufficient to improve the mechanical properties of bioprinted structures undergoing degradation. The initial phase of the interaction of the biological material with the hydrogel is mediated by proteins present on the cell surface, such as integrins, syndecans, and cell adhesion molecules. Integrin plays a key role here by binding to cell-adhesin motifs of the hydrogel, which regulates the downstream mechanotransduction pathway and affects the mechanical properties of the hydrogel. Figure 8: Results of the evaluation of the changes in the mechanical properties of bioprinted hydrogel structures after their individual cultivation periods. The team of [PERSON] et al. describes that typically, in three-dimensional hydrogel systems, cells with a diameter of about 1 \(\upmu\)m are embedded in a hydrogel with a pore size of 10-100 nm. This proportion indicates that degradation of the hydrogel matrix is necessary to allow the migration and even spreading of cells in the 3D matrix. This process directly affects the mechanical stability of the hydrogel network [47]. The highest, equal to 1.78 \(\pm\) 0.87 N, average value of tensile strength of the bioprinted structures was registered after a cultivation time of 6 weeks. The dynamic increase in the samples' maximum breaking force is the result of an intense homogeneous overgrowth of the printout structures by the biological material contained in them after a prolonged time of incubation. SEM observations and live/dead assay results confirm that fibroblast cells during this period form a durable, homogeneous, highly branched network within the material structure, mimicking the tissue structure. In 3D cultivation, released proteins accumulate near the cells instead of diffusing freely into the culture medium. Furthermore, numerous growth factors are associated with ECM proteins, which direct proliferation and morphogenesis in three-dimensional cultures [71]. The research conducted by the team of [PERSON] et al. confirms the thesis that in the case of tubular vascular printouts, their tensile strength increases with the cultivation time of the structures, during which the production and organization of ECM by biological material takes place [72].