Methods : AECs were isolated from 3 months sheep. Cell morphology and stem cell markers expression were analyzed by FACS at different passages of in vitro expansion. Osteogenic differentiation was induced in 6thpassage cells. Differentiated AECs (d-AECs) were analyzed at fluorescence and light microscope. Osteogenic differentiation was also checked by RT-PCR evaluation of bone tissue related molecules. Critical bone defects in sheep were used for in vivo testing the regenerative potential of d-AECs; after 45 days, animals were euthanized and block sections obtained for radiographic and microscope evaluation.
Results: FACS showed AECs positivity for typical surface markers. Culture passage had moderate effects on cellular conditions, as some adhesion molecules expression gradually decreased. With increasing number of culture passages, Sox2 and TERT expression decreased, while NANOG one increased. Interestingly, cultured AECs tended to aggregate into tridimensional spheroides. Increased ALP activity, OCN and COL1 expression in d-AECs compared to undifferentiated cells confirmed the occurrence of osteogenic differentiation. Moreover, d-AECs showed a marked calcein deposition and ECM mineralization, as revealed by fluorescence and light microscope. Histological analysis of block sections comprising d-AECs graft, permit to identify abundant new bone formation in continuity with the host bone tissue. Fluorescence microscope analysis showed AECs were still recognizable in the grafted sites after a 45 days healing period.
Conclusions: In vitro results confirm the elevated AECs potential to differentiate into multiple cell lines, and to undergo rapid and extensive in vitro osteogenic differentiation. Moreover, in vivo results reveal the ability of d-AECs to induce bone regeneration in critical size defects, also showing AECs may maintain stemness characteristics for medium long period.