# Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth
## 2 Materials and Methods
### Evaluation of Rheological Properties of Polymer Solutions  
The rheological properties of the 2A9G polymer composition prepared as described in Section 2.3., containing no biological material, and a bioink containing 1\(\cdot\)10\({}^{6}\) fibroblast cells/mL were tested using an MCR702 Multidrug rheometer (Anton Paar GmbH, North Ryde, Australia). The polymer solutions were placed on the lower measurement plate of the device, and the gap between the measuring plates was set to 0.5 mm, using the geometry of a parallel plate of diameter 25 mm. The measurements were taken at a temperature of 34 \({}^{\circ}\)C, during which the 3D printing process takes place. The viscosity was measured in the shear rate speed range of 0.1 to 1000 1/s. Before each measurement, the polymer solutions underwent thermostating for 10 min with the purpose of obtaining an even polymer solution temperature distribution over the volume of the material. Assessment of the Viability of Fibroblast Cells of the NIH/3T3 Line Contained in Hydrogel Printouts after Individual Periods of Cultivation of Structures Using the Fluorescent Live/Dead Assay  
The analysis of the viability of the fibroblast cells contained in hydrogel printouts was carried out with live/dead staining using a mixture of dyes, with calcein-AM applied for the green labeling of live cells and ethidium homodimer III (EthD) used for the red labeling of dead cells. The cells incorporated in bioinks and hydrogel printouts were imaged using a fluorescent microscope (Nikon Eclipse LV100 ND, Tokyo, Japan). The images of live and dead cells were taken in 10 randomly chosen areas of the analyzed samples. Three samples of tubular printouts were assigned for each of the cultivation time points. Based on recorded images, the number of live and dead cells was calculated using ImageJ 1.8.0 (LOCI, University of Wisconsin, WI, USA). To determine the cell viability of the NIH/3T3 line, the following relationship was used:  
\[\textit{Viability}\;[\%]=\frac{\textit{the number of living cells}}{\textit{the number of living cells}+\textit{the number of dead cells}}\cdot\] 100% (1)  
The results presented in the following sections show the calculated average viability of the cells incorporated in the hydrogel printouts. Figure 2: Schematic of the preparation of hydrogel bioinks and 3D bioprinted structures. Source: elaborated by authors using BioRender.com [46]. Evaluation of the Proliferation of Fibroblast Cells of the NIH/3T3 Line Embedded inside the Printouts after Individual Periods of Cultivation of the Structures Using a Scanning Electron Microscope (SEM)  
The bioprinted hydrogel structures were fixed with a 2.5% glutaraldehyde solution at a temperature of 4 \({}^{\circ}\)C for 12 h before the SEM observation process. Subsequently, the hydrogel materials were rinsed twice with PBS and subjected to a dehydration process with successively increasing concentrations of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) for 10 min. The dehydrated printouts were dried in a dryer at the critical point in the LEICA EM CPD300 (Leica, Wetzlar, Germany). Finally, the samples were coated with a 5-7 nm thick conductive layer of carbon fiber using a sputtering unit SafeMatic CCU-010 LV Compact Coating Unit (SafeMatic, Zizers, Switzerland). After the applied preparation, the degree of proliferation and morphology of fibroblast cells incorporated inside the printouts after individual periods of culture of the structures were evaluated using a scanning electron microscope JSM-6610 LV (Jeol, Akishima, Tokyo, Japan). Evaluation of the Mechanical Properties of Bioprinted Hydrogel Structures Subjected to Individual Cultivation Periods Using a Static Tensile Test  
The mechanical properties of the bioprinted hydrogel structures were evaluated using a static tensile test with a holder adapted to the specific geometry of the samples (Figure 3), which held the tubular printout in a stable position during the tensile test and allowed the samples to be placed in the holder without first damaging their structure.