BASIC SCIENCE & PATHOLOGY

THE EFFECT OF VITAMIN D ON VASCULAR ENDOTHELIAL GROWTH FACTOR IN BONE

M Horgan, I D McCarthy
Department of Orthopaedic Surgery, Imperial College School of Medicine, Charing Cross Campus, Fulham Palace Road, London W6 8RF, UK

Angiogenesis is critical for successful osteogenesis during endochondral ossification and fracture repair. The endothelial cell-specific mitogen Vascular Endothelial Growth Factor (VEGF) is an essential mediator of angiogenesis and is expressed by hypertrophic chondrocytes in the epiphyseal growth plate. It has already been demonstrated that human osteoblasts produce VEGF, the steady-state level of which is stimulated by Vitamin D in its active form 91,25-Dihydroxycholecalciferol. The study set out to investigate whether or not Vitamin D supplementation showed any increase in the production and action of VEGF in the bony epiphysis and whether there was a corresponding increase in bone proliferation at the same site. A study was undertaken on the proximal tibiae of Wistar rats divided into three groups: one group fed on a diet with normal levels of Vitamin D, the second group fed on a diet with low supplementation of Vitamin D, and the third group was fed on a diet with high-level supplementation of Vitamin D. The animals were sacrificed at six weeks of age and the proximal tibiae removed from each one. The left tibiae were randomly divided to two halves, one of which was placed in EDTA for decalcification, the other half being snap-frozen in liquid nitrogen and held in a freezer at -80 degrees, both sets of bones awaiting histology, immunohistochemistry or VEGF assay. The right proximal tibia of each animal was placed in formalin for basic morphometry measurements.

Aortic blood samples were taken from each animal and blood Vitamin D was measured via Competitive Protein-binding Assay, and levels reflected the relative Vitamin D intake of each animal according to the supplementation received. VEGF in the epiphysis was quantitatively determined using VEGF immunoassay, which confirmed the presence of VEGF in bone and the quantity present in the samples of each group. The lengths and weights of each bone showed a trend whereby greater lengths and weights were seen in the control groups as opposed to the supplemented groups.