| Fracture repair is a reproducible cascade of cellular events that leads to bony union of the broken fragments. After a fracture , the space between the ends of the broken bones is filled with hematoma rich in pleuripotential mesenchymal cells and cytokines. The tissue that forms within this space ( callus) usually goes through a cascade of events that including cartilaginous , ossification and remodeling.NO is a free radical gas which has been implicated as a secondary messenger molecule in many biological pathways. NO is generated by the conversion of the basic amino acid, L - arginine to L - citrulline in a reaction catalysed by a family of isoenzymes arising from three different genes. They are eNOS , iNOS and bNOS. NO takes part in many pathophysiological process in human bodies. Its functions in orthopaedics include inflammation, arthritis, osteoporosis, aseptic loosening, even in fracture healing.Our aim is to evaluate the expression of NOS in the process of fracture healing. It is our hypothesis that NO production would affect the process of fracture healing in femoral fractured model of rats by o-ral L - NAME and L - arginine.Materials1. Establishment of fracture model60 Wistar rats were divided into 3 groups randomly. Each group had 20 rats to include the 2 test group and 1 control group. The rats in testing group drank water with L - NAME (0.05% ) and L - arginine (0.4% ) , while the rats in control group drank water only. One day before fracture , all the animals started to drink the above fluid . In the anesthesis of 0.1% chloroaminoketone we build the closed femoral fracture model.2. Selection of specimen3 days , 1 week, 2 week and 4 week after the fracture, 5 rats in each group were killed to get the specimen. 1cm callus in the fractured femur and the same in the normal contralateral femur were selected in this step. The specimens were fixed in 10% formalin solution and decalcified in the equal quantity mixed solution of 8 mol/L formic acid and 1 mol/L formic sodium. The specimens were embedded in paraffin and sectioned longitudinally, dehydrated in a series of alcohol, been hyaline in dimethylbenzene. All the specimens were observed by HE stain and immunohistochemical methods.Methods1. HE stain was used to evaluate the healing degree of fracture according to the proportion of fibroblast, chondroblast, woven bone tissues and mature bone tissues. There were 10 grades all together.2. Immunohistochemical methods( SP method)The slices were removed wax to hydrate first; 3% H202 was applied to block the activity of endogenous peroxidase; digested by complicated digestive solution and sealed by serum. Then were dropped with the first antibody NOS(iNOS, eNOS and bNOS) overnight and the biotin - labelled second antibody; streptoaffinin peroxidase; DAB colored; ended by the water; restained by hematoxylin; differentiated by 1 % hydrochloric alcohol; washed in flow water and dehydrated and been hyaline. PBS solution were used between these steps and at the last, observed in the microscope for the expression of NOS.3. Analysis of results:The results of HE stain were made a table to analyze by the Student -1 test.ResultsNo NOS expression in the contralateral femur and bNOS expression in the fractured femur were seen. The expression of iNOS was not the same as that of eNOS, iNOS was expressed all through the process of fracture healing; while eNOS was later than iNOS . They had different intensity of expression. NOS were expressed mainly in the fibro-blast, chondroblast and the endothelium of vascules.Compared to the control group, the fracture in L - NAME group had a slower healing speed (p =0.10) , while L - arg had no significant difference with it. Compared the two test group, L - arg had a faster healing velocity than that of L - NAME( P < 0.05 ).DiscussionsNO was found and acknowleged since 1980s and was looked as an important messenger and effecting molecule. Its functions in many pathophysiological process had been clarified gradually. The...
|
PDF Full Text Request