Increased bioactivity in fluoride and high phosphate containing glasses and stimulation of VEGF production in MC3T3-E1 osteoblast-like cells

Objectives: Optimal bone regeneration is dependent on sufficient blood supply; bioactive glasses (BG) could act as a delivery system to promote local vasculogenesis. Phosphate plays an important role in BG apatite formation and fluoride (F^-) stimulates osteoblast activity in vitro. Vascular endothelial growth factor (VEGF) is important for the regulation of vasculogenesis and angiogenesis. The aim of this study was to investigate bioactivity and VEGF producing potential in osteoblast-like cells of high P₂O₅ containing bioactive glasses with low F^- levels.
Methods: BG (0-7% F^- content, and 6.33% P₂O₅ in mole%) were produced by a melt-quench method and then ground and sieved to <38µm. Glass powder was immersed in Tris Buffer solution (1.5g/L) for 2h, 8h, 24h, 72h and 168h to determine apatite formation through X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Ion (Ca, P, Si and F) release by dissolution was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) and fluoride-selective electrode. RT-PCR and Western Blot were used to quantitate the effects of 72 hour glass-conditioned medium on VEGF gene and protein expression in MC3T3-E1 cells.
Results: XRD and FTIR demonstrate an amorphous glass structure in all groups (0-7% F^-). Apatite formation was accelerated in the F^- added group (even 1% content) after only 2-8h immersion in Tris buffer. Apatite was not found in the F^- free group until 72h immersion. Ca, P and F release reached the peak after 2h immersion and then decreased. VEGF gene expression was increased, and protein expression dose-dependently promoted with media conditioned by F^- containing glasses from 1 to 21 days in culture.
Conclusions: The combination of a low F^- content with high phosphate significantly accelerated apatite formation in Tris Buffer. F^- containing glass conditioned medium significantly promoted VEGF production in MC3T3-E1 cells. F^- containing BGs might accelerate optimal bone regeneration.