Session III - Polytrauma Fracture Healing

Fracture Healing is Modulated by Nitric Oxide

Ashish Dhar Diwan, MD; Min-Xia Wang; George A. C. Murrell, MD, Orthopaedic Research Institute, St. George Hospital, UNSW, Sydney, Australia

Fracture healing is a well regulated cascade of cellular events. Regulation of the cellular events in fracture healing are not clearly understood. The role of the messenger molecule nitric oxide (NO) in fracture healing has not been studied. A group of enzymes called the nitric oxide synthases (NOS) catalyse the conversion of L-arginine to nitric oxide and L-citrulline. Nitric oxide synthase isoforms are classed as constitutive (Ca⁺⁺ dependent) or inducible (Ca⁺⁺ independent). The end products of NO metabolism are nitrate (NO₃^-) and nitrite (NO₂^-).

Purpose: The first part of this study aimed to identify in a temporal fashion the presence of nitrates and nitric oxide synthases in rat femoral fracture repair. The second part of the study was designed to determine if there was a modulatory role for NO in rat fracture repair. The final part of the study evaluated the presence of NO^. in human fracture callus.

Methods: Study I: 32 male Sprague Dawley rats had a right femoral fracture created by three-point bending. Rats were sacrificed on day 4(n=7), 7(n=6), 10(n=6), 15(n=6), and day 30(n=7). The control group consisted of weight matched (n=6) rats without any fracture. Nitrate was measured in the serum of these rats by gas chromatography mass spectrometry. Fracture callus NOS activity was quantified using a ³H L-arginine to ³H L-citrulline conversion assay. Polymerase Chain Reaction (PCR) and Western Blot were carried out to identify the mRNA and protein of iNOS in the fracture callus respectively. Immunohistochemistry was performed to localise iNOS protein. Study IIA: 30 male Sprague Dawley rats had an open right femoral midshaft fracture created and fixed with a K-wire. The treatment group(n=13) had a nitric oxide inhibitor, L-NAME (1mg/ml) added to their drinking water ad lib. Treatment was administered preoperatively and then for 21 days, at the end of which all rats were sacrificed. Observers blinded to the groups measured callus diameter, bone mineral density and three- point bending. Study IIB: was designed to evaluate the role of a nitric oxide donor in fracture healing. 48 male SD rats had an open right femoral midshaft fracture created and fixed with a K-wire. A carboxybutyl compound of the polysaccharide chitosan (CBC) was used to deliver nitric oxide locally at fracture site. Rats were divided into four groups viz. L-NAME (n=13), D-NAME an inactive enantiomer of L-NAME (n=13), CBC-NO (n=11) and vehicle alone CBC (n=11). Blinded observers measured callus diameter. Study III: Fresh fracture callus was obtained from patients undergoing surgery four days or more after trauma. The presence and/or absence of NOS was evaluated by Western blot, PCR and immunohistology.

Results: Study I: The mean(SD) serum nitrite concentration prefracture was 21(3)nmol/ml. Two peaks in serum nitrate post fracture were on day seven 36(4) (p<0.001) and day thirty 38(17)nmol/ml (p<0.001). Ca⁺⁺ independent NOS activity was absent in the unfractured group but peaked on day 15 (p<0.05). Ca⁺⁺dependent constitutive NOS activity was absent prior to fracture and gradually increased to 1.9 pmol/mg prot (p<0.01) on day 30. A strong signal for mRNA of iNOS was detected by RT-PCR on day 15. The protein for iNOS was detected at all stages of fracture healing but not in the controls. Immunoperoxidase staining for iNOS on day 10 of fracture showed staining of cartilage like cells. Study II: Rats fed the NOS inhibitor L-NAME had a 20% reduction in callus circumference (p<0.001), 20% reduction in bone mineral density(p=0.08) and 30% reduction in peak loads on three point bending (p<0.05). Callus morphometry results in study IIB revealed a 30% (p<0.01) increase in callus size in the NO donor group compared to the NOS inhibited group. Study III: The protein and mRNA for NOS were identified in human fracture callus.

Conclusions: These results show for the first time that NO^. is present during fracture repair in rats and humans. Inhibition of NO synthesis in rats reduces fracture diameter, mineral density and strength, while addition of NO enhances fracture healing.