Article
Sildenafil stimulates angiogenesis and bone formation in a non-union model in mice
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Published: | October 25, 2022 |
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Objectives: Although the knowledge of fracture healing and orthopedic fracture care improved markedly during the last decades, delayed healing and non-union formation remain a major clinical problem. Segmental bone defects caused by high-energy trauma, infections and tumor are particularly associated with a high rate of atrophic non-union formation. Non-unions are considered to be the result of an avascular and biologically inert environment. Hence, new therapy options need to stimulate angiogenesis and other biological processes to improve fracture healing. Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phosphodiesterase-5-inhibitor, has become the most widely used drug for erectile dysfunction in men. Moreover, several studies reported that Sildenafil exerts pro-angiogenic and pro-osteogenic properties. Therefore, we investigated whether sildenafil stimulates bone formation and improves bone repair in a non-union model in mice.
Methods: In 12-14 weeks old CD-1 mice a 1.8mm femoral segmental defect was created and stabilized with a custom-made pin-clip osteosynthesis. The animals were treated with a daily dose of 5 mg/kg body weight sildenafil p.o., controls received equivalent amounts of vehicle. Two, five and ten weeks after the creation of the segmental defect, the process of bone regeneration was analyzed by X-ray, biomechanics, photoacoustic imaging, µ-CT, histology, histomorphometry and Western blot analysis. Values are expressed as mean ± SEM. Statistical significance: P < 0.05.
Results and conclusion: Sildenafil-treated animals showed a significantly increased bending stiffness at 10 weeks after surgery (1.74 ± 0.39 vs. 0.51 ± 0.09 N/mm), when compared to controls. This was associated with an enhanced oxygen saturation (40.71 ± 5.88 vs. 11.84 ± 4.33 %) and bone tissue formation (5.03 ± 0.75 vs. 1.60 ± 0.21 mm³). Moreover, the histological analysis revealed an increased number of TRAP-positive osteoclasts (13.65 ± 1.89 vs. 6,43 ± 0,79 osteoclasts/(high power field (HPF)) and CD31-positive microvessels (20.75 ± 0.63 vs. 16.26 ± 1.33 microvessels/HPF) within the callus tissue of sildenafil-treated mice. In addition, Western blot analysis revealed an elevated expression of the pro-angiogenic and pro-osteogenic factors cysteine-rich protein 61 (6.71 ± 3.63 vs. 3.19 ± 2.03 pixel intensity 104) and VEGF (30.20 ± 11.15 vs. 10.83 ± 2.30 pixel intensity 104), confirming the increase of bone formation and angiogenesis.
These findings indicate that sildenafil stimulates angiogenesis and bone regeneration in an atrophic non-union model in mice by increasing the expression of the pro-angiogenic and pro-osteogenic markers CYR61 and VEGF, inducing the infiltration of macrophages to the fracture site and delaying the process of bone resorption. Therefore, sildenafil may be a promising compound in the treatment of non-union formation in clinical practice.