| BackgroundFracture healing is a complicated and precisely regulated process. For the events leading to it, three important ingredients are required: 1)cells playing roles in bone formation;2)osteoinductive factors;3)an environment in which osteoinduction is sustained.In orthopaedic folklore an accelerated rate of fracture healing has long been associated with traumatic brain injury. However, the underlying mechanism is still unknown. And establishing appropriate animal models and Exploring the mechanism may provide further avenues for the treatment of both non-union and heterotopic bone formation. Semaphorin 3 A is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated Sema3A regulates osteogenesis directly by acting on osteoblasts and osteoclasts[1], and indirectly by influencing innervation[2]. Our team had revealed there was a change of Sema3A concentration in serum from mice with traumatic brain injury and femoral fracture and its effect on osteoclasts’ differentiation and function,however,we still don’t know whether fracture healing is accelerated when with traumatic brain injury and whether Sema3A’s effect on innervation plays a role in it. So we establish rat models with traumatic brain injury and femoral fracture, observe fracture healing with multiple techniques, detect the change of Sema3A concentration in sera, observe the distribution of nerve fibres in the fracture region to find out what role Sema3 A play in fracture healing when with traumatic brain injury.ObjectiveTo establish standard rat models which are subjected to closed femoral fracture and traumatic brain injury, and to detect the change of Semaphorin 3A concentration in serum by ELISA and the expression of GAP43/PGP9.5 in the fracture region by immunohistochemisty, and to find out more about the mechanism by which accelerated fracture healing is associated with traumatic brain injury.Methods① A weight-drop device was made to establish models of traumatic brain injury in rats. And at the same time, models of standard closed femoral fracture were established using an apparatus designed by us. Vital signs of rats were observed after injury immediately. At 6 hours after injury, magnetic resonance imaging(MRI) scan was performed to make sure that if models of traumatic brain injury had been successfully constructed, and the Revised Neurobehavioral Severity Scale(NSS-R)was used to evaluate the extent of rats’ brain injury at 24 hours post injury. At 7 days after injury, the pathological changes of brains were observed. At 1,2,4 weeks after injury, X-ray pictures of left femur were taken to assess fracture healing, and at 2,4,8 weeks after injury, left femurs were harvested, and Micro-CT scan of them was performed to assess callus formation,② Adult male Sprague-Dawley rats were divided into two groups: Group 1, only fracture; Group 2, traumatic brain injury with femoral fracture. At 5 days, 7days, 14 days, 21 days after injury, sera were harvested.③ ELISA analysis was used to detect the expression of Semaphorin 3 A in sera.④ At 2 weeks after injury, the expression of GAP43(marker for regenerating nerve fibers), PGP9.5(markers for mature nerve fibers) in the fracture region was detected by immunohistochemisty to observe the distribution of nerve fibers.Results① After traumatic brain injury, the rats showed noticeable changes of vital signs which included apnea, oronasal bleeding, and so on. And MRI showed edema and intracranial hematoma. And remarkable functional changes of central nervous system were observed by NSS-R. Gross observation showed subarachnoid hemorrhage, scars in brain. X-ray test revealed that short oblique or tranverse fractures in femurs.Micro-CT showed microstructure characteristics of callus at each time point.Histologically, all the fractures healed by secondary intention.② X-ray tests, Micro-CT scans, and histological tests revealed excessive callus formation and a faster fracture healing in rats with traumatic brain injury and femoral fracture.③ Sema3A concentration in rats with traumatic brain injury and femoral fracture was higher than rats with femoral fracture only at 7d, 14d, 21d after injury(p<0.05).④ At 2 weeks after injury, there were more GAP43-positive and PGP9.5-positive nerves in the fracture region in rats with traumatic brain injury and femoral fracture than in rats with femoral fracture only.Conclusion① A highly reproducible rat model with traumatic brain injury and femoral fracture is established ,which can be used in next experiments.② Sema3A concentration of sera after traumatic brain injury increased significantly,And Sema3A influences innervation, there were more nerve fibers in the fracture region in rats with traumatic brain injury and femoral fracture. And Sema3A may be associated with excessive callus formation and accelerated fracture healing by influencing innervations. |
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