Results 26 evaluated from passage 1 upwards. Cells in normoxic and hypoxic in vitro environment demonstrated a similar proliferation up to day 12. Subsequential, normoxic cell proliferation decreased constantly until the population went into senescence after 52 days in culture and reached a cum PD of 15.79 (Fig.10, red dashed line). In contrast, hypoxic cultured cells showed a consistent, more homogeneous and faster proliferation up to 52 days and went as recently as 64 days after extraction into senescence 64 days after extraction (Fig.10, blue dashed line). The cum PD was almost doubled with 28.49 compared to the cells in 21% oxygen. An illustration of the entire time of evaluation is presented in figure 10. Fig. 10: Cumulative population doublings (cum PD) of normoxic and hypoxic cultured hMSCs. Up to day 12 cells showed a comparable proliferation. The following days, normoxic cultured cells decreased in proliferation and went into senescence after day 52 (red dashed line). Hypoxic cultured cells showed a nearly linear proliferation over the entire time period until they went into senescence after day 64 (blue dashed line). Additional information of in vitro culturing of cells can be obtained with the calculation of the population doubling time (PDT). In comparison to cum PD, the PDT offers a direct insight into the replication time. During the first 12 days, with comparable results for the cum PD of normoxic and hypoxic cultured cells, the PDT showed 44.66 ± 10.23 h for normoxic cells and 38.19 ± 7.33 h for hypoxic cells, respectively. Despite the homogenous cumulative population doublings, the (PDT) showed a rapid changing over the different passages for the normoxic group. A complete evaluation showed a mean PDT of 100.90 ± 53.52 h for normoxic cells and 72.90 ± 72.69 h for hypoxic cultured cells over the entire period. The large standard deviation for cells in
Results 27 Hypoxia demonstrated the prolonged plateau phase during cum PD. The exclusion of the three last senescent passages of hypoxic cultured stem cells reduced the mean PDT to 47.98 ± 11.08 h. Figure 11 shows an overview of the PDT trend. Fig. 11: Population doubling time (PDT) of normoxic and hypoxic cultured hMSCs. Cells cultured under standard conditions showed a highly inhomogeneous PDT until day 40 and did not increase their dividing time before senescence. A superior homogenous PDT was detected for hypoxic cells, while a high increase during senescence occurred. Dashed lines indicate the beginning of population senescence. 3.2.2. Metabolic activity Another component of cellular proliferation is the metabolic activity of intracellular mitochondria. Therefore, cellular metabolism was measured with a WST-Assay over passage 4 to 6. Normoxic cultured hMSCs showed a mean relative absorbance of 100.0 ± 3.2%, whereas hypoxic cells showed a significant higher metabolic activity (136.9 ± 37.05%, p=0.0012) compared to the normoxic control (Fig. 12).