Nitric oxide in fracture repair - Differential localisation, expression and activity of nitric oxide synthases

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, re sponsible for the generation of NO, are expressed during the healing of fra ctures. To localise the sites of expression compared with those in normal b one we made standardised, stabilised, unilateral tibial fractures in male W istar rats. Immunostaining was used to determine the precise tissue localis ation of the different NOS isoforms, Western blotting was used to assess ex pression of NOS isoform protein and L-citrulline assays for studies on NOS activity, Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the earl y phase of fracture repair. Western blot and image analysis confirmed this initial increase, Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture h ealing. Western blotting showed a reduction in iNOS during the early healin g period. Significantly reduced calcium-independent NOS activity was also s een. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blo od flow recognised during fracture healing. eNOS expression in osteocytes m ay occur in response to changes in either mechanical or local fluid shear s tress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.