Background: In high-risk patients up to 45% of fractures heal inadequately, resulting in nonunion development with major consequences for patients quality of life. To enhance bone healing, sufficient production of nitric oxide (NO), solely derived during the conversion of arginine into citrulline by nitric oxide synthases (NOSs), may be essential. NO stimulates bone cells to regulate bone remodelling, influences vascular reactivity and enhances collagen synthesis, all essential components of normal fracture healing.
Methods: In 20-24 week old wild type and either inducible or endothelial NOS (respectively NOS2 or NOS3) deficient mice (n=8/group), periosteal cauterisation and a 0.45 mm femur osteotomy was performed. Callus volume was measured using micro-computed tomography (μCT) after 7, 28 and 42 days of fracture healing. Myeloperoxidase was measured with immunohistochemistry to determine neutrophil influx in callus tissue and bone marrow. Femurs were also sampled for RNA analysis of relevant enzymes and high-performance liquid chromatography measurement of amino acids.
Results: After 28 and 42 days of fracture healing, NOS deficient animals showed evident nonunion, while union was reached in all wild type animals after 28 days. Both NOS knock out groups showed an increased neutrophil influx compared with wild type mice both at 1 week and 1 month of fracture healing. At these time points, amino acid concentrations of arginine, ornithine and citrulline, and expression of enzymes related to the arginine-NO metabolism were deregulated in NOS deficient animals when compared to wild type mice.
Conclusion: In the present study we showed for the first time that the absence of either NOS3 or NOS2 results in depleted substrate metabolism and an inadequate fracture healing. Enhanced myeloperoxidase levels indicate a role of disturbed inflammatory response in the development of non-union in NOS depleted animals.
Disclosure: The authors declared no competing interests.