Growth and Co-culture of Endothelial Cells on Ordered Fluorapatite Surfaces

Fluorapatite (FA) crystal has been previously shown to be able to stimulate the differentiation of human adipose derived stem cells (ADSCs).  Objectives: To study the effect of ordered FA crystal on the growth of endothelial cells and on the mineralization of co-culture of endothelial cells and ADSCs.  Methods: Human microvascular endothelial cells (HMVECs) were subcultured in EGM-2-MV medium on the FA surfaces and etched stainless steel (SSE) surfaces for 1-28 days. The initial cell adhesion and cell growth up to 14 days were examined. The specimens were also observed under SEM and analyzed by flow cytometry. The releasing of fibroblast growth factor-2 (FGF-2) from the cells grown on the experimental surfaces was detected by enzyme-linked immunosorbent assay (ELISA). The HMVECs were then co-cultured with ADSCs and stained with Alizarin red after 28 days.  Results: There was no significant difference of cells initially attached to and grown on the two surfaces after 1, 7 and 14 days. Consistently, the cell cycle analysis at day 3 showed similar level of the proliferation indexes (PrI, S+G₂M) of cells on the two surfaces. SEM showed that the HMVECs cells spread well and flattened with more extensions on FA surfaces. A significant higher amount of FGF-2 was detected in cells grown on FA surfaces after 1 and 7 days. Interestingly, the mineral nodule formation was observed in the co-cultures grown on the two surfaces even without the osteogenesis induction (OI), with much stronger Alizarin red staining on the FA surfaces than that on SSE surfaces.  Conclusions: FA surfaces showed good biocompatibility and supported the growth of endothelial cells. The secretion of soluble growth factors like FGF-2 from the endothelial cells and their intimate interaction with ADSCs may play important roles in stimulating the mineralization of the co-cultures grown on the FA surfaces.