## 3 Results
### Ingrowing of Stromal Cells Into Decellularized Tissue and Its Differentiation Towards SMCs  
An analysis of the histological images (demonstrated in Figure 7) and the cell numbers at given depths (demonstrated in Figure 8) shows a fundamental difference between static cultivation and  
Figure 6: Evaluation of the decellularization process using cryosections 10 μm in thickness stained with DAPI. The brightfield images and the fluorescence images were combined to visualize both the tissue and the remaining nuclei/DNA. From top to bottom: A sample of native tissue with visible cell nuclei. The tissue after SDS cycles, where no cell nuclei are visible, but the residual DNA is split over the tissue. A completely decellularized tissue with no remaining visible cells or DNA residues. The Leica DMi 8 microscope, 10x objective, and scale bar 100 μm were used.  
dynamic cultivation. In static conditions, there is no significant difference between 5 and 10 days of cultivation. In both cases, the cells penetrated to a depth of 50 \(\upmu\)m, but there is no evidence of cell ingrowth into deeper regions of the tissue.  
Figure 7: DAPI counterstained composed of recellularized tissues cultivated in static and dynamic conditions for 5 days and for 10 days. Tissues prior to cultivation were seeded from a single side of decellularized tissue. The microscope used Leica DMi 8, 10\(\times\) objective.  
However, the results for dynamic cultivation show improved penetration of the cells into all layers of the tissue, with some decreasing character where the number of cells decreases with depth. At depths \(>\)100 \(\upmu\)m, there is a significant difference between static cultivation and dynamic cultivation. In both time intervals, there are observable cells in all layers of the dynamically-cultivated samples. After 10 days of dynamic cultivation, it can be seen that almost all the tissue has been newly recellularized with cells.  
An analysis of the growth and differentiation of cells on the surface of the decellularized matrix showed that there is a major difference between static cultivation and dynamic cultivation after 10 days of cultivation. As shown in Figure 9, under static conditions, the cells form clusters instead of spreading homogeneously over the entire surface. The signal from h1-calponin is also weaker and is present in a smaller number of cells. Under dynamic conditions, however, a homogeneous cell layer is formed, and h1-calponin is present in most of the cells, including cells with well-developed calponin fibres. In this case, increased cell growth and overall improved growth of cells on the surface of decellularized tissue can also be observed under dynamic culture conditions.  
Figure 8: The number of cells in given depths of tissue in static and dynamic conditions with single side seeding. Statistical significance is for \(p\) < 0.05. Ten samples were taken for every type of cultivation and time interval. Each of the samples was sliced to 50 \(\upmu\)m depth regions where cell numbers were counted. Red lines represent the median values; blue boxes 1 st and 3 rd quartiles; whiskers are the minimum and maximum; and the red circles are outliers.