| IntroductionWith the development of the traffic, high energy trauma,caused by car accidents,could result in serious combined injuries, such as comminuted fractures,or multiple fractures, serious sofe tissue injuries etc. All of the combined injuries would result in bony defect,lack of blood supply and poor mechanical stability, which can increase the chance of nonunion. So far there are many ways of treating the nouunion, such as extracorporeal shock wave, autogenous bone graft etc. All ways were to recover limb function, rectify deformity and improve living quality.Mechanical stress stimulation has a strong correlation with fracture union.Many literatures of animal and clinical studies have proved the mechanical load has a critical role in maintaining the structure and morphology of bone trabecular. Wolff found that trabecular was mainly distributed along the direction of principle stress and the strength of bone in this direction was superior to the surrounding area. Weight bearing-activities could also improve bone quality and osteoporosis. The express osteogenic cytokines as BMP, VEGF and TGFβ can also be elevated under the intermittent mechanical stress, which can promote the surrounding vascular remodeling process and increase blood supply for bone union.Based on these background data, we raised a hypothesis that intermittent mechanical stress could promote the proliferation and differentiation of osteogenic cells surrounding non-union site, stimulate the expression of cytokines and finally reached successful union. In this study we built femoral diaphyseal non-union model in rats and used the axial pressure effect of external fixation to provide mechanical stress. The bone microstructure and bone quality as well as the changes of cytokines as BMP, VEGF and TGFβwere measured to discuss the relative factors during the development and healing of non-union. The results showed that mechanical stress was a helpful stimulus to non-union healing, which provided valuable background for further studies.Methods1. The development of femur diaphyseal nonunion modelFemale Sprague-Dawley rats at6months of age were used to build nonunion animal,which was anesthetizedwith chloral hydrate (0.3ml/100g) intraperitoneal injection, and a single hindlimb was shaved and was prepared Iodophor. A sterile technique was used at all times.Transverse incision was used at the middle part of femur. The femur was exposed with blunt dissection and the periosteum was preserved. Four0.99mm transverse-threadedKirschner wires was used to drill holes first among lesser trochanter and trochanter tertius, then from distal to proximal direction in proper order.A6-mm critical-sized full-thickness defect was created in the diaphysis with use of a1-mm side-cutting burr under irrigation with saline solution. External fixation was applied onto Kirschner wires and to provide constant pressure on the defect. The surroundingmuscle cuff and skin were closed in order. Penicillin was administrated intramuscular postoperatively for3days. The animals were monitored daily for another12weeks, after which they were killed for specimen collection. X-rays were taken to observe the bone formation across the defect. Three independent, blinded observers scored the radiographs with use of a previously described5-point scale to assess the percentage of bone formation across the defect. Sagittal cuts containing the proximal and distal host-defectinterfaces were embedded in polymethylmethacrylate and surface-stained withHematein-Eosin stain.2. The effect of mechanical loading on the model of femur diaphyseal nonunionThe external fixation was designed to adjust the defect thickness with the block2and3. The thickness was shortened about2mm in the first month. Food stimulus was used to induce rats doing high-intensity activities about10minutes as a cycle. Ten cycles were repeated every day with30minutes intermittent among them. In the next months, the defect thickness was further shortened2mm every month in the same method. In the control group, the defect thickness remained6mm unchanged during3months. X-rays were taken to observe the bone formation across the defect at the end of12weeks. Three independent, blinded observers scored the radiographswith use of a previously described5-point scale to assess thepercentage of bone formation across the defect. Sagittal cuts containing the proximal and distal host-defectinterfaces were embedded in polymethylmethacrylate and immunohistochemical assay was used to detect the level of according osteogenic cytokines.Results1. The development of femur diaphyseal nonunion model1.1Radiographic resultIn experiment group there was no observable callus formation in the defect at week0,2,4,6,8,10and12week. At week12, X-ray showed that the bone marrow cavity of both sides were blocked osseous tissue and remained at same level. The external fixation remained stable throughout the study.1.2Hematein Eosin stainAt week2, few nucleus dark stained osteogenic cells were found around defects, which are surrounded by large amount of inflammation cells. At week4, the number of osteogenic cells increased and some cartilage cells were observed. At week8, the osteogenic cells were rarely seen, replaced by large number of abundant chondroid cells and spindle oval fibroblast cells. At week10, large amounts of woven bone tissue could be observed. At week12, the bone marrow cavity of both sides was blocked osseous bone, surrounded by few osteogenic cells and large amount of spindle fibroblast cells.2. The effect of mechanical loading on the model of femur diaphyseal nonunion2.1Radiographic resultNo callus was formed at week4,8and12weeks, the thickness of defects remained unchanged and both ends showed pestle-like shape. In experimental group, the defects were obscure in X-ray picture with the shortening of thickness. Subperiosteum ossification could be observed around wire holes. At week8, cotton-like shadow could be observed in the defects. In some samples, newly formed bone tissue could be found around unilateral cortex. At week12, the defects were shorted obviously and obscure; obvious new bone formation could be observed, some samples showed unilateral bony connection.The radiographic score of experimental group was significantly higher than that in control group from week4(p<0.05). The score of experimental group at week12was significantly higher than the score at week4and week8(p<0.05).2.2Immunohistochemical assayIn control group, low level expression of BMP, VEFG and TGFβ could be detected at week4,8and12. However, it remained low level throughout the study without peak value observed.The local expression of BMP was significantly elevated at week4compared to base value (p<0.05). The value reached peak value at week8, which was significantly higher than that at week4and week12. The expression value of week12was reduced, but it was still significantly higher compared to control group (p<0.05).The peak value of local expression of VEGF reached peak value early at week4, which was significantly higher than base value, week8and week12(p<0.05). There were no significant difference between the value at week8and week12, which were all significantly higher than control group (p<0.05).The value of local expression of TGFβ was significantly higher than control group throughout the study (p<0.05). However, no significant difference could be detected among the value at week4,8and12.3. Calcium cobalt stainThe number of osteoblast began to elevate at week4, which was significantly higher than control group. The value kept arising at week8and reached peak value compared to week4and12(p<0.05). In control group, low level of osteoblast was detected at week4,8and12. However, it remained low level throughout the study without peak value observed.Conclusion1. There was low level of osteogenic cytokines expression in the local environmentof non-union site. Mechanical stress could elevate the expression of osteogenic cytokines around the site of non-union.2. The bony connection caused by mechanical stress could be confirmed from radiographic and histomophology assays.Key Innovation1. The study confirmed that there was continuous low level of expression at non-union local environment.2. The study confirmed that mechanical stress could elevate the expression of osteogenic cytokines at non-union local environment and provided the fundamental theory for further study about the effect of mechanical stress on non-union. |
