## 4 Discussion
Cyclic physiological pressure (CP) of 120/80 torr altered the cellular morphology and increased the proliferation rates in stem cells over a 7-day period as described in the study by [PERSON] et al. [28]. Moreover, dynamic seeding using a bioreactor enables preconditioning of the cells under more physiological conditions, as described in the study by [PERSON] et al. [15]. A custom dynamic cultivation system built in our laboratory was used in this study. This system generates a microperfusion flow and pulsatile hydrostatic pressure. As mentioned above, in static conditions, the cells penetrated only in 50 \(\upmu\)m depth. The tissues cultivated in dynamic conditions proved significantly better penetration rates of cells. Significant results are observable from 100 \(\upmu\)m depths. At depths \(>\)150 \(\upmu\)m and more, there is nearly no evidence of cells ingrowing in static conditions. As is illustrated in Figures 7 and 8 after 10 days of dynamic cultivation, most of the tissue has been newly recellularized with cells. As was mentioned, we tried to demonstrate the efficiency of a dynamic cultivation bioreactor in stimulating cell ingrowth inside the decellularized tissue. So, the technique of seeding utilized only one side of the decellularized tissue. Thus, the number of cells decreased with the penetration depth. To promote more homogenous ingrowing of cells, the cultivation chamber was optimized for dual side seeding of cells. In this approach, both sides of decellularized tissues were seeded with the same density of cells prior to cultivation. As is illustrated in Figures 11 and 12, the cells are homogeneously distributed inside the recellularized tissue. Also, the time period used to achieve this was reduced to 5 days. Also, the differentiation of ASCs towards the SMC phenotype can be achieved via biochemical and mechanical stimulation. A positive effect on the differentiation of stem cells towards SMCs has been demonstrated in studies using a combination of TGF-8 and BMP4 growth factors. This led to an increase in the production of specific cytoskeletal protein markers of differentiation, such as \(\upalpha\)-actin and h1-calonin, which are present only in differentiated SMCs [29], after 7 and more days of static cultivation. Cell differentiation towards the SMC phenotype can be further enhanced by appropriate mechanical stimulation in a dynamic cell culture system. The positive effect of mechanical stimulation on differentiation towards SMCs has been described for cells cultured either with or without the presence of TGF-8 and BMP4 [17; 30]. In addition, mechanical stimulation helps to keep the cells in a differentiated state and prevents de-differentiation [31]. As for mechanical stimulation, we applied physiological hemodynamic force, which is stimulation with cyclic hydrostatic pressure (CP) 120/80 mmHg. The CP induced differentiation of the seeded adipose-derived stromal cells towards the SMC phenotype. This was proven by the expression of an SMC marker calponin. Greater cell population density and greater upregulation of early differentiation markers \(\alpha\)-actin and calponin were achieved in a pressure-stimulated culture than in static conditions [18; 32]. In this context, two mechanosensitive signaling pathways associated with SMC differentiation have been described: the RhoA-associated pathway, and the FAK kinase-associated pathway. Activation of these signaling pathways leads to increased synthesis of SMC differentiation markers in the cells [33]. 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.