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Objectives and questions: Non-unions remain among the most serious complications in trauma surgery. The expression profile of pro-angiogenic and pro-osteogenic growth factors at the fracture site plays a central role in bone healing. Adequate vascularization is crucial for supplying the callus tissue with nutrients, oxygen and stem cells.Moreover, the expression of vascular endothelial growth factor (VEGF) receptors may influence angiogenesis at the fracture site and, consequently, the healing process. However, the exact expression profiles of angiogenic and osteogenic growth factors as well as VEGF receptors between unions and non-unions remain unknown.

Material and methods: Growth factor and VEGF receptor expression were investigated during physiological fracture healing and in non-unions of the femora in 2–4 months-old CD1 mice. For physiological fracture healing, an intramedullary screw was used as a model for a closed, stable fracture (union group). To generate non-unions, a 1.8 mm segmental defect was created and stabilized with an intramedullary pin. For additional rotational stability, the defect was further stabilized with a ventro-dorsal clip (non-union group). Femora were harvested after 7, 10, and 14 days, as well as 10 weeks post-surgery. The callus tissue was investigated by X-ray, µCT, biomechanical testing, histology and Western blot analyses. Data are given as Mean ± SEM.

Results: Biomechanical, histological, and radiological analyses demonstrated reliable fracture healing in the union group, whereas in the non-union group, no bony bridging of the fracture gap was observed in any of the animals up to 10 weeks after surgery. Western blot analysis revealed a significantly higher expression of the VEGF2 receptor in non-unions when compared to unions 14 days after surgery (1.7 ± 0.5 vs. 14.9 ± 6.8 [pixel intensity*104]). Moreover, the ratio of VEGF2 receptor/VEGF (0.04 ± 0.01 vs. 0.69 ± 0.35) was significantly higher, whereas the ratio of VEGF1/VEGF2 receptor (29.97 ± 10.30 vs. 1.87 ± 0.75) was significanlty lower. Interestingly, the overall expression of the blood vessel marker CD31 did not differ between the two groups.Additional analyses demonstrated a significantly reduced expression of bone morphogenetic protein (BMP)2 in non-unions 7 days after surgery.

Discussion and conclusions: These novel data demonstrate significantly altered expression profiles of VEGF receptor as well as pro-angiogenic and pro-osteogenic growth factors in unions and non-unions. The increased expression of the VEGF2 receptor along with elevated VEGFR2/VEGFR1 and VEGFR2/VEGF ratios indicate an overshooting pro-angiogenic signaling, which may result in uncoordinated vascularization and a dysfunctional vascular network, ultimately hampering bone regeneration. In addition, the reduced expression of the pro-osteogenic markers BMP2 and CYR61 at an early healing time point may contribute to non-union formation. By specifically targeting these dysregulations, novel therapeutics may overcome bone healing failure.