Temporal and spatial vascularization patterns of unions and nonunions: role of vascular endothelial growth factor and bone morphogenetic proteins.

ABSTRACT

BACKGROUND: Failure of fracture-healing with nonunion is a major clinical problem. Angiogenesis is closely linked to bone regeneration, but the role of angiogenesis in nonunion formation remains unclear. Because established nonunions are well vascularized, we hypothesized that lack of vascular endothelial growth factor (VEGF) expression and vascularization during the early time course of fracture-healing determine nonunion formation. METHODS: In seventy-two CD-1 mice, a femoral osteotomy with a gap size of 1.80 mm (nonunion group) or a gap size of 0.25 mm (union group) was created and stabilized by a pin-clip technique. Healing was analyzed after three, seven, fourteen, twenty-one, twenty-eight, and seventy days by micro-computed tomography and histomorphometry. Vascularization was determined in different healing zones by immunohistochemical staining of PECAM-1 (platelet-endothelial cell adhesion molecule). Additional animals were analyzed after seven, fourteen, and twenty-one days with Western blot analysis of VEGF, bone morphogenetic protein (BMP)-2, and BMP-4 expression. RESULTS: Micro-computed tomography and histomorphometry showed complete bone-bridging in the union group, whereas animals in the nonunion group showed atrophic nonunion formation. Vascularization increased from day 3 to day 7 in both groups, with a subsequent decrease after fourteen days. However, overall vascularization did not differ between unions and nonunions over time. It is of interest that vascularization within the endosteal healing zone was even higher in nonunions than in unions after fourteen days. Expression of VEGF was significantly higher in nonunions, while expression of BMP-2 and 4 and proliferating cell nuclear antigen were found significantly reduced compared with unions. CONCLUSIONS: Because vascularization during the early time course of fracture-healing was not impaired despite the failure of bone-healing in nonunions, we rejected our hypothesis and accepted the null hypothesis that nonunion formation is not due to failure of VEGF-mediated angiogenesis. Failure of fracture-healing was associated with a decreased expression of BMP-2 and 4 and a disturbed ratio of angiogenic to osteogenic growth factors, which may be responsible for nonunion. CLINICAL RELEVANCE Because the intrinsic angiogenic response during nonunion formation was sufficient for adequate vascularization, treatment strategies for nonunions should focus on the stimulation of osteogenesis rather than on the stimulation of angiogenesis.