3.1. Alizarin Red S staining of osteogenic differentiated hBMSCs
After 21 d of osteogenic differentiation hBMSCs were treated with different concentrations of VP and TXA (VP3TX10, VP12TX10, VP50TX10, VP3TX50, VP12TX50, VP50TX50) for 24 h and 48 h to examine the effects of combined treatment of TXA and VP on matrix mineralization at these two exemplary chosen time points. After this treatment Alizarin Red S staining was performed on exemplary cultures treated with the lowest (VP3TXA10) and highest (VP50TXA50) concentration combinations for 24 h and 48 h, to examine osteogenesis in differentiated hBMSCs (Fig. 1, a). Untreated (Fig. 1, a, no treatm.) and undifferentiated negative controls (Fig. 1, a, undiff.) were maintained for comparison.
Undifferentiated control samples showed no positive Alizarin Red S staining (Fig. 1, a, undiff.) while all osteogenically differentiated samples showed positive Alizarin Red S staining after 21 d independent of the exposure time and treatment with VP and TXA (Fig. 1, a, no treatm., 24 h, 48 h).
Furthermore, no visible changes regarding the Alizarin Red S staining intensity could be observed in dependence of the varying concentration combinations of VP and TXA and the examined exposure time, in osteogenic differentiated hBMSCs derived from all five patient samples (Fig. 1, a, 24 h, 48 h). When comparing cell cultures treated with VP and TXA to untreated control cultures and undifferentiated cultures no alteration of cell size was observed (Fig. 1, a)
The calculation of the Alizarin Red S standard curve revealed a high coefficient of determination R2 (Fig. 1, b). Quantifications of Alizarin Red S staining showed low concentrations of Alizarin Red S in undifferentiated cultures (Fig. 1, c, undiff.) in comparison to osteogenic differentiated cultures independent of treatment with VP and TXA (Fig. 1, c, control, VP3TX10, VP50TXA50). Although there was a non-significant trend towards lower Alizarin Red S concentrations in cultures treated with rising concentrations of VP and TXA (Fig. 1, c, VP3TXA10, VP50TXA50) compared to untreated control groups (Fig. 1, c, control), this trend was non-significant.
3.1. ATP Assays of osteogenic differentiated hBMSCs
hBMSCs were seeded in monolayer cultures and were exposed to an osteogenic differentiated medium. Following osteogenic differentiation for 21 d cells were exposed to different concentration combinations of TXA and VP (VP3TX10, VP12TX10, VP50TX10, VP3TX50, VP12TX50, VP50TX50) for 2 h, 24 h, 48 h and 96 h. We examined the effects of the combined treatment with VP and TXA on cell proliferation rates using the ATP Assay (Fig. 2), while untreated osteogenic differentiated cultures were maintained as negative control groups (Fig. 2, control). Bar charts were used to show the mean values with the corresponding standard deviations.
Following 2 h of treatment with varying concentration combinations of TXA and VP no effects on proliferation rate in monolayer cultures was observed in comparison to control cultures (Fig. 1, 2h). After increasing exposure time to 24 h proliferation rate was lowered significantly in cultures treated with VP50TX10 (Fig. 2, 24h, VP50TX10). In addition, in cultures treated for 24 h there was a non-significant trend towards a lower proliferation rate in almost all other cultures (Fig. 2, 24h).
Following 48 h and 96 h of exposure to varying concentration combinations of VP and TXA there was a clear trend towards a dose and exposure time-dependent effect on proliferation rate in examined cultures (Fig. 2, 24h, 48h, 96h). Proliferation rates in comparison to untreated control groups were significantly lower in cultures exposed to concentration combinations containing 50 mg/mL of VP after 48 h (Fig. 2, 48h, VP50TX10, VP50TX50). After 96 h of exposure to VP and TXA significant reductions in proliferation rate were also visible in all other cultures exposed to VP and TXA, except those treated with the lowest concentration combination of VP3TX10 (Fig. 2, 96h).
3. 2. Annexin 5 assays of osteogenic differentiated hBMSCs
Cell viability and apoptosis of osteogenic differentiated MSC cultures, derived from two randomly chosen donors, following treatment with different concentration combinations of VP and TXA (VP3TX10, VP12TX10, VP50TX10, VP3TX50, VP12TX50, VP50TX50) was examined using double fluorescence staining with Annexin 5 (dead cells) -Cy3/6-carboxyfluorescein diacetate (living cells) (Fig. 3). Untreated cultures were used as negative controls (Fig. 3, untreated controls). Exposure time varied between 2 h, 24 h, 48 h and 96 h.
The control groups as well as osteogenic differentiated cultures treated with different combinations of VP and TXA for up to 24 h mostly showed a distinct green fluorescence marking viable cells (Fig. 2, untreated controls, 2 h, 24 h).
Although viable cells were also visible in cultures treated with VP and TXA for at least 48 h (Fig. 3, 48h, 96h), these were reduced in number. In addition, the apoptosis detection assay revealed that cultures treated with varying combinations of VP and TXA for 48 h or 96 h reacted more heavily Annexin 5-positive than control cultures or those treated for a maximum of 24 h, resulting in a clear red fluorescence (Fig. 2, 48h, 96h).
When comparing cultures treated for 48 h, there was also a trend towards less green, viable cells and a more intense red staining in osteogenic differentiated cultures treated with higher concentrations of VP (Fig. 3, 48h, VP50TXA10, VP50TXA50).
The control groups as well as cultures treated with different combinations of VP and TXA for up to 24 h mostly showed a distinct green fluorescence marking viable cells (Fig. 2, untreated controls, 2 h, 24 h).
Although viable cells were also visible in cultures treated with VP and TXA for at least 48 h (Fig. 3, 48h, 96h), these were clearly reduced in number. In addition, the apoptosis detection assay revealed that cultures treated with varying combinations of VP and TXA for 48 h or 96 h reacted more heavily Annexin 5-positive than control cultures or those treated for a maximum of 24 h, resulting in a clear red fluorescence (Fig. 2, 48h, 96h).
When comparing cultures treated for 48 h, there was also a trend towards less green, viable cells and a more intense red staining in osteogenic differentiated cultures treated with higher concentrations of VP (Fig. 3, 48h, VP50TXA10, VP50TXA50).
3. 4. Expression of osteogenic marker genes
RT-PCR was performed to evaluate changes in the relative expression of osteogenic marker genes in osteogenic differentiated monolayer cultures derived from five different donors (n = 5) after treatment with different combinations of VP and TXA (VP3TX10, VP12TX10, VP50TX10, VP3TX50, VP12TX50, VP50TX50) for varying exposure times (2 h, 24 h, 48 h, 96 h) in comparison to untreated negative controls (Fig. 4).
There was no significant impact of treatment with varying concentration combinations of VP and TXA on the expression of examined osteogenic marker genes COL1A2, COL10A1, ALP and OC in cultures independent of the assessed exposure time (Fig. 4).
However, there was a non-significant trend towards a declining relative expression of the osteogenic marker genes COL1A2, COL10A1 and ALP depending on the exposure time (Fig. 4, a-c). This non-significant decline of the relative expression of respective osteogenic marker genes was evident after 48 h, continued over the exposure period of 96 h and was independent of examined concentration combinations (Fig. 4, a-c, 48 h, 96 h). This non-significant exposure time-dependent effect of VP and TXA on the relative expression of osteogenic marker genes was not observed for OC (Fig. 4, d).
In addition, no clear differences regarding the impact of varying concentration combinations on the relative expression of osteogenic marker genes was observed for the examined exposure times (Fig. 4).