title: Deficiency of inducible and endothelial nitric oxide synthase results in diminished bone formation and delayed union and nonunion development.
abstract: BACKGROUND
  Between 5% and 10% of all fractures fail to heal adequately resulting in nonunion of the fracture fragments. This can significantly decrease a patient's quality of life and create associated psychosocial and socio-economic problems. Nitric oxide (NO) and nitric oxide synthases (NOS) have been found to be involved in fracture healing, but until now it is not known if disturbances in these mechanisms play a role in nonunion and delayed union development. In this study, we explored the role of endothelial and inducible NOS deficiency in a delayed union model in mice.
  
  
  METHODS
  A 0.45mm femur osteotomy with periosteal cauterization followed by plate-screw osteosynthesis was performed in the left leg of 20-24week old wild type, Nos2(-/-) and Nos3(-/-) mice. Contralateral unfractured legs were used as a control. Callus volume was measured using micro-computed tomography (μCT) after 28 and 42days of fracture healing. Immuno histochemical myeloperoxidase (MPO) staining was performed on paraffin embedded sections to assess neutrophil influx in callus tissue and surrounding proximal and distal marrow cavities of the femur. After 7 and 28days of fracture healing, femurs were collected for amino acid and RNA analysis to study arginine-NO metabolism.
  
  
  RESULTS
  With μCT, delayed union was observed in wild type animals, whereas in both Nos2(-/-) and Nos3(-/-) mice nonunion development was evident. Both knock-out strains also showed a significantly increased influx of MPO when compared with wild type mice. Concentrations of amino acids and expression of enzymes related to the arginine-NO metabolism were aberrant in NOS deficient mice when compared to contralateral control femurs and wild type samples.
  
  
  CONCLUSIONS
  In the present study we show for the first time that the absence of nitric oxide synthases results in a disturbed arginine-NO metabolism and inadequate fracture healing with the transition of delayed union into a nonunion in mice after a femur osteotomy. Based on these data we suggest that the arginine-NO metabolism may play a role in the prevention of delayed unions and nonunions.
authors.affiliation: Department of Surgery and Trauma surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; NUTRIM School for Nutrition and Translational Research in Metabolism, PO Box 616, 6200 MD, Maastricht, The Netherlands. Electronic address: d.meesters@maastrichtuniversity.nl.
authors.forename: D M
authors.initials: DM
authors.lastname: Meesters
authors.affiliation: AO Research Institute Davos, Clavadelerstrasse 8, 7270, Davos, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Neubert
authors.affiliation: Department of Surgery and Trauma surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; NUTRIM School for Nutrition and Translational Research in Metabolism, PO Box 616, 6200 MD, Maastricht, The Netherlands.
authors.forename: K A P
authors.initials: KAP
authors.lastname: Wijnands
authors.affiliation: Department of Surgery and Trauma surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; NUTRIM School for Nutrition and Translational Research in Metabolism, PO Box 616, 6200 MD, Maastricht, The Netherlands.
authors.forename: F L
authors.initials: FL
authors.lastname: Heyer
authors.affiliation: AO Research Institute Davos, Clavadelerstrasse 8, 7270, Davos, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Zeiter
authors.affiliation: AO Research Institute Davos, Clavadelerstrasse 8, 7270, Davos, Switzerland.
authors.forename: K
authors.initials: K
authors.lastname: Ito
authors.affiliation: Department of Surgery and Trauma surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands.
authors.forename: P R G
authors.initials: PRG
authors.lastname: Brink
authors.affiliation: Department of Surgery and Trauma surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; NUTRIM School for Nutrition and Translational Research in Metabolism, PO Box 616, 6200 MD, Maastricht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Poeze
mesh_terms: D000596:Amino Acids / Q000097:blood; D000818:Animals; D001119:Arginase / Q000235:genetics / Q000378:metabolism; D002146:Bony Callus / Q000000981:diagnostic imaging / Q000201:enzymology / Q000473:pathology; D005260:Female; D005599:Fractures, Ununited / Q000097:blood / Q000000981:diagnostic imaging / Q000201:enzymology* / Q000473:pathology*; D008810:Mice, Inbred C57BL; D052247:Nitric Oxide Synthase Type II / Q000172:deficiency / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency* / Q000378:metabolism; D010012:Osteogenesis; D009195:Peroxidase / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D055114:X-Ray Microtomography
chemical_list: D000596:Amino Acids; D012333:RNA, Messenger; D009195:Peroxidase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D001119:Arginase
keywords: Arginine; Delayed union; Fracture healing; Nitric oxide; Nonunion