Intrinsic Osteogenic Potential Of Sheep Endothelial Progenitor Cells

Objectives: In-vivo bone formation potential by transplanted EPC's was shown in previous studies, suggesting that EPC's have intrinsic osteogenic potential. The aim of this study was to examine the in-vitro osteogenic potential and characteristics of sheep EPC's grown in osteogenic differentiation medium, compared with control medium (non-osteogenic medium).
Methods: Mononuclear fraction was isolated from sheep peripheral blood. The mononuclear fraction was seeded and grown in conditions that promote EPC culture (EBM2 medium). Cells were grown at 37^oC with humidified 95% air 5% CO₂. Non-adherent cells were discarded by gentle washing with PBS every 3 days. Following expansion, cells were divided into two groups: test group was grown in osteogenic differentiation medium: EBM2 containing 10% FCS, Dexamethasone 10-7 M, ascorbic acid 5x10-5 M and β-glycerophosphate 10-2 M, control group was grown in EBM2-medium. Alkaline phosphatase (AP) activity was examined two weeks and three weeks after cell population division. AP Activity was detected by using BCIP/NBT substrate solution.
Results: Three weeks following isolation, the tested cell population presented cellular characteristics of "late EPC" including: unique cobblestone morphology, high proliferation rate and expressed endothelial markers. Following 2-3 weeks of culture in EGM-2 or in osteogenic medium, osteogenic markers such as AP activity and polygonal morphology were identified. Unexpectedly, about 5 percent of the cells were osteogenic transformed without significant differences between the groups. Osteogenic differentiated cells were stained positively for BCIP/NBT, presented polygonal morphology and larger cytoplasm compared with non-differentiated negatively stained cells that were round. Moreover, proliferation rate was decreased over time in both groups.
Conclusions: EPC's have intrinsic osteogenic potential, and can spontaneously transform in-vitro into osteogenic cells, regardless their growth environment.