# 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 performed analysis showed that after 8 weeks of cultivation of the samples, their tensile strength was significantly reduced to 0.18 \(\pm\) 0.03 N. Obtaining tensile properties close to the initial cultivation times of bioprinted samples is most likely the result of a very strong cellular overgrowth of tubular structures, which can be characterized by local inhomogeneities in the thickness of the cellular network with complete degradation of the polymer matrix at the same time. In addition, during the 8-week incubation, the results of the SEM observations confirm a change in cell morphology from the spindle shape characteristic of fibroblasts spanning the polymeric structure to a spherical morphology. This phenomenon may be a result of the dense packing of biological material in the printout structure, leading to limited space for further cell growth and spreading. The high density of biological material also leads to more cell-to-cell interactions and, thus, increased metabolic activity and the intense expression of collagen, the accumulation of which can act to increase the rigidity of the constructs and, thus, result in their loss of elasticity [73]. These observations may result in the high brittleness of hydrogel samples that have been completely overgrown with biological material, in the structure of which degradation of the polymer matrix has occurred initially due to the thermal dissociation of gelatin and subsequent gradual decross-linking of the alginate network as a result of the ion exchange that is occurring. This process can also be accelerated due to the need for regular changes in the culture medium of the bioprinted structures, which intensifies the exchange rate of calcium ions cross-linking the hydrogel with sodium ions present in the culture medium environment. The observed decrease in tensile strength of printouts subjected to two months of incubation may also be a result of a stress gradient caused by the presence of cellular contraction forces, which can cause mechanical instability and deformation of the structure. Indeed, there is a correlation between cell density, the concentration of hydrogel material, its degradation, and changes occurring in the structure of the printouts caused by forces generated by growing cell networks, which can induce buckling, axial contraction, failure, or total static stability. The biological material dynamically interacts with the surrounding polymer matrix and generates traction forces that influence the macromolecules of the degrading hydrogel. The cells form focal adhesions with the ECM, and transmembrane integrins transmit mechanical force [52; 74; 75]. The literature data confirm that the mechanical properties of such structures can change over time together with changes in the density and distribution of biological material [52]. After 8 weeks of bioprint culturing, there can also be a complete imbalance in the system in terms of the calcium metabolism. This phenomenon occurs due to the constant supply of new growth medium, which causes a succession of ion exchanges, resulting in decalcification of the cells and leading to disintegration of their cell membrane, which results in the observedmorphological changes and the disappearance of the mechanical properties provided by a coherent network of cell matrices. In addition to signaling pathways, ion channels such as Ca\({}^{2+}\) are an important part of membrane polarity. The flow of Ca\({}^{2+}\) ions through ion channels and transporters induces changes in the cytoskeleton [76]. The regulation of calcium ion concentrations is crucial for the proper functioning of cells, because calcium performs a role in almost all cellular processes [77]. In addition, the changes in extracellular osmolarity that occur as a result of continuous exchange of growth media can lead to changes in cell volume and, therefore, in the intracellular concentrations of macromolecules, which is a key physical parameter affecting the spatial organization of cells [78].