The effect of bisphosphonates on the endothelial differentiation of mesenchymal stem cells

Abstract

The contribution of the local stem cell niche to providing an adequate vascular framework during healing cannot be overemphasized. Bisphosphonates (BPs) are known to have a direct effect on the local vasculature, but their effect on progenitor cell differentiation is unknown. This in vitro study evaluated the effect(s) of various BPs on the differentiation of human placental mesenchymal stem cells (pMSCs) along the endothelial lineage and their subsequent functional and morphogenic capabilities. pMSC multipotency was confirmed by successful differentiation into cells of both the osteogenic and endothelial lineages, as demonstrated by positive Alizarin Red S staining and Ac-LDL uptake. pMSC differentiation in the presence of non-cytotoxic BP concentrations showed that nitrogen containing BPs had a significant inhibitory effect on cell migration and endothelial marker gene expression, as well as compromised endothelial differentiation as demonstrated using von Willebrand factor immunofluorescence staining and tube formation assay. This in vitro study demonstrated that at non-cytotoxic levels, nitrogen-containing BPs inhibit differentiation of pMSCs into cells of an endothelial lineage and affect the downstream functional capability of these cells supporting a multi-modal effect of BPs on angiogenesis as pathogenic mechanism contributing to bone healing disorders such as bisphosphonate related osteonecrosis of the jaws (BRONJ).

Figure 1

(a,b) The effect of bisphosphonates on pMSC proliferation. 2 μM CLO, 1 μM ALN and 0.5 μM ZA were considered to be optimal concentrations for the differentiation studies without adversely affecting cell survival over a 10-day period.

Figure 2. Live/Dead^® analysis of pMSCs cultured in BP containing media.

At the optimal BP concentrations: CLO 2 μM, ALN 1 μM and ZA 0.5 μM, as suggested by the cell proliferation study (Fig. 1), significantly more live cells (and fewer dead cells) were observed after 10 days of culture.

Figure 3. Light microscope images show in vitro wound healing after 6 hrs and 24 hrs was significantly inhibited in the presence of BPs.

Furthermore, quantitative analysis (histogram) showed that inhibition of wound healing by the higher potency ALN (at 6 hrs) and ZA (at both 6 and 24 hours) were both significantly higher (p < 0.0001) than with the lower potency CLO.

Figure 4

Osteogenic differentiation capability of (a) pMSCs. Following culture in osteogenic media for 2 weeks (b), the cells showed significant Alizarin Red S staining of a mineralised cell matrix (c).

Figure 5. Endothelial lineage of the VEGF treated pMSCs was confirmed by Ac-LDL uptake after 10 days.

(a) Control pMSCs, (b) Endothelial differentiated pMSCs and (c) Endothelial differentiated pMSCs showing uptake of fluorescent labelled Ac-LDL.

Figure 6. pMSCs differentiation into endothelial cells as assessed by the intensity of Von Willebrand factor staining was inhibited by both ALN and ZA.

Compared to unstimulated pMSCs, CLO had a minimal effect on vWF expression. vWF staining in HUVECs was included as a positive control. Legend: DAPI (4′,6-Diamidino-2-Phenylindole); cell nuclear stain, Anti-vWF; FITC labelled Von Willebrand factor, Merged; combined DAPI and anti-vWF staining, HUVEC; human umbilical vein endothelial cells, EGMV; endothelial growth media supplemented with VEGF

Figure 7. Wimasis WimTube (Wimasis GmbH Munich, Germany) generated image showing significantly reduced tube formation by pMSC derived endothelial cells in the presence of BPs compared to both stimulated pMSCs and HUVECs.

Figure 8. Quantitative analysis of the morphological tube formation parameters; total covered area, total loop length, loop numbers and the number of branching points (Wimasis WimTube, Wimasis GmbH Munich, Germany) were all significantly reduced in the pMSC derived endothelial cells in the presence of BPs compared to both stimulated pMSCs and HUVECs.

Inhibition was potency dependent with the most significant inhibition seen in the ALN and ZA treated cells. (Significant differences, compared to EGMV are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Figure 9. Relative gene expression of stem (CD73 and CD105) and endothelial (von Willebrand Factor, PECAM-1, VEGF-R1, VEGF-R2, and CD34) cell markers in pMSCs after 1, 5 and 10 days differentiation in EGMV alone or with the BPs CLO (2 μM), ALN (1 μM) or ZA (0.5 μM).

Significant differences in expression compared to EGMV are indicated as *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.0001.