Abstract
Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.
Highlights
Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair
Diabetes patients suffer from impaired bone formation and fracture healing[17], which may be a consequence of diabetes, related cardiovascular disease, which restricts blood supply to the bone[18]
Treatment of human osteoblast-like MG-63 cells with 25 ng/mL or 100 ng/mL rhVEGF165 protein for 24 hours resulted in a significant increase (116.4% and 116.0% respectively; P < 0.0001) in DNA content compared to non-treated controls as measured using the PicoGreen assay
Summary
Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascular endothelial growth factor (VEGF) was first shown over two decades ago to be the key factor coupling osteogenesis and angiogenesis as inactivation of VEGF concomitantly subdued blood vessel invasion and bone formation[7,8]. VEGF receptors including VEGFR1 (Flt1), VEGFR2 (Kdr), and VEGFR3 (Flt-4) are expressed by both blood vessel forming endothelial cells[9] and bone forming osteoblasts[10,11]. Current treatments are limited and often require invasive surgery, with long recovery times and can be dependent upon the available vasculature within bone grafts[21,22,23] As these pathologies and morbidities are associated with a lack of an adequate functional bone vasculature, a suitable approach for treatment necessitates methods of controlling and enhancing angiogenesis in bone. In an osteoblast specific Vegfa knockout mouse model, the absence of osteoblast-derived VEGF was linked to decreased intramembranous bone formation, decreased angiogenesis and a reduction in callus remodelling as assessed using a tibial bone drill defect model[29]
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