# Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth
## 2 Materials and Methods
### Preparation of Hydrogel Bioink and 3D Bioprinted Structures  
For the preparation of 3D bioprinted structures, a composition of hydrogel materials based on alginic acid sodium salt from brown algae and gelatin from porcine skin type B was used. The hydrogel solution was prepared in the DMEM high-glucose culture medium. Before the preparation process of hydrogel solutions, appropriate amounts of sodium alginate (A) and gelatin (G) powders underwent UV radiation sterilization for 1 h. The polymer solution containing 2% sodium alginate and 9% gelatin (2A9G) was prepared in a weight-volume ratio by mixing the alginate and gelatin together in such a way that it was possible to \"subtract\" from the mixture of 8% culture medium that had previously been added to the cell pellet before combining it with the hydrogel. The polymer solution was mixed at a temperature not exceeding 40 \({}^{\circ}\)C at a speed of 200 rpm for 2 h. The missing amount of medium, equal to 8% of the solvent volume used to prepare the hydrogel, was then added to the flask containing the NIH/3T3 cell pellet. An appropriate amount of hydrogel providing a seeding density equal to 1\(\cdot\)10\({}^{6}\) cells/mL was added to the cell suspension. The cell suspension was combined with the hydrogel using a piston pipette. The mixing time did not exceed 1 min. The prepared hydrogel bioink was placed in a sterile syringe with a volume of 5 mL and subjected to the 3D bioprinting process of tubular structures with a length of 30 mm and an inner diameter of 2.8 mm (Figure 1). The 3D printing process was performed using a 3D printer designed and constructed at the Technical University of Lodz. The 3D printing process, during which the material is pneumatically extruded, was performed on a horizontal shaft using a conical nozzle. The height of the print layer was set at 220 um. The 3D printing process was conducted at a temperature of 34 \({}^{\circ}\)C.  
A 1% solution of calcium chloride prepared in saline was used to cross-link the printouts. The cross-linking time of each of the samples was 10 min. The cross-linking process was performed at room temperature. After the structures were cross-linked, each sample was rinsed three times with PBS buffer. The bioprinted structures underwent cultivation on 6-well plates in DMEM high-glucose culture medium supplemented with 10% CBS and 1% P/S/N under standard conditions for the following individual time periods: 0, 1, 4, 6, and 8 weeks. Each of the incubation periods was a separate measurement system. A schematic of the preparation of bioprinted structures is shown in Figure 2. During cultivation, the culture medium was changed every 2-3 days. The multi-well plates on which the bioprinted structures were cultivated were replaced once a week by transferring the samples to the surface of new multi-well plates.  
Bioprinted structures after individual cultivation periods were subjected to assessment of the viability and proliferation of fibroblast cells contained in the hydrogel samples using a fluorescent live/dead assay, an analysis of the changes in the cell morphology using a scanning electron microscope (SEM), and an assessment of mechanical properties using a static tensile test.  
Figure 1: (**A**) Schematic of a 3D bioprinted structure. (**B**) Image of 3D bioprinted structure directly after the cross-linking processes.  
Figure 2: \(Cont\).