Objectives: 

There are several studies about bone fracture union and bone transplantation but the biology of cells transplanted within bone grafts is not completely understood. The aim of this work is improve the knowledge of in vivo angiogeneic regulation during bone grafting thinking in medical applications.

Materials: 

We report a mouse femur graft model in which grafts were derived from mice transgenic for a Firefly luciferase (FLuc) bioluminescence reporter gene driven by a promoter for the angiogenic signaling molecule vascular endothelial growth factor A (VEGF-A). Results were analyzed with in vivo non-invasive bioluminiscence imaging (BLI) and bone histology. Primary graft-derived osteoblastic cells (GDOst) from femur and skull were characterized and used for cell culture studies, together with other bone-related cell lines (7F2, MC3T3-E1#4, MC3T3-E1#24 and CH3T1/2). RNA levels were measured with reverse transciptase polymerase chain reaction (RT-PCR) and protein level with enzyme-linked immunosorbent assay (ELISA).

Results: 

Upon transplantation into wild type (wt) mice, in vivo BLI permitted longitudinal visualization and measurements of VEGF-A promoter activity in the transplanted graft cells and demonstrated a lag period of 7 days post transplantation prior to robust induction of the promoter. To determine cellular mediators of VEGF-A induction in graft bone, primary GDOst were characterized. Cell culture experiments and BLI analysis on GDOst showed hypoxia induced VEGF-A expression, and that this induction depended on PI3K signaling and to a lesser degree on the MEK pathway. This transcriptional regulation correlated with VEGF-A protein production and was validated in GDOst demineralized bone matrix (DBM).

Conclusions: 

Non invasive BLI of VEGF-A is feasible in bone tissue and is useful to study bone gaft biology. Together, combined imaging of VEGF-A expression in living animals and in living cells provided clues about the regulation of angiogenesis in bone grafting. This data is significant towards the development of future smart bone graft substitutes.