| Objective To observe the effects of stromal cell-derived factor-1(SDF-1),vascular endothelial growth factor(VEGF)and the combined effects of two growth factors during femoral distraction osteogenesis in rats.The aim of this study was to explore the mechanism of DO and find a new strategy for shortening the treatment time of DO.Methods Ninety-six male Sprague-Dawley rats were randomized into four groups of 24 rats each.The left femoral DO model was established.All the rats were performed osteotomy in the middle of the left femurs.After osteotomy,the left femur was fixed by external fixator.After a latency period of 5 days,gradual distraction was performed at a rate of 0.25mm per 12 hours for 10 days,resulting in a total of 5 mm lengthening.Growth factors were injected directly into the distraction gap on seven consecutive days after completing distraction.Group A was injected with 1ml PBS vehicle;Group B was injected with 100ng SDF-1 in 1 ml PBS solution;Group C was injected with 50ng VEGF in 1 ml PBS solution;Group D was injected with 100ng SDF-1 and 50ng VEGF in 1 ml PBS solution.(1)At the 2nd?the 4th?the 6th and the 8th week after the cessation of distraction,six rats were randomly selected to observe the progress of bone formation through X-ray.(2)At the 4th week after the cessation of distraction,Six rats were randomly selected from every group,and the progress of bone formation was determined with microcomputed tomography(micro-CT).Three-dimensional images were reconstructed and the bone volume(BV),bone mineral density(BMD)and trabecular thickness(Tb.Th)were measured in the new bone.(3)The osteoblast-related genes were detected by RT-PCR:Runx-2?OPN?ALP and OCN.(4)At the 4th and the 8th week after the cessation of distraction,six rats were randomly selected from every group and HE stain was performed to observe the histological structure.(5)Three-point bending biomechanical tests were performed on the femurs after performing the Micro-CT biomechanical tests.Results(1)Radiographically In the second week after the cessation of distraction,none of the group A rat femurs showed callus in the central zone of the distraction gap.There is a small amount of new bone formed on both group B and group C,and the formation of extra callus was more obvious in group B than group C.New bone formation was observed obviously in Group D,and the stretching gap was reduced significantly.In the fourth week after the cessation of distraction,there was obvious new bone formation in group A,and the osteotomy line was visible clearly.The stretching gap in group C almost disappeared and the osteotomy line was obscure.In group B and group D,the stretching gap exhibited bridging callus formation,but the density of callus in group B was lower than that in group D.In the six week after the cessation of distraction,the stretching gap of rat femurs in group B,group C and group D all demonstrated bone connection,whereas the osteotomy line of rat femurs in group A was still observed.All groups rats femurs exhibited bridging callus formation 8 weeks after cessation of distraction,and the new bone remodeling of group D is better than the other three groups.(2)For micro-CT,bone formation and remodeling of the distraction regeneration in group D had greater values than others groups at 4 weeks after the cessation of distraction.Two-dimensional quantitative analysis of the distraction regeneration showed that the bone volume of group D had higher values than the other three groups(P<0.05).(3)RT-PCR test The expressions of osteoblast-related genes such as Runx-2,OPN,ALP and OCN in group D were significantly higher than those in other groups(P<0.05).There was difference between group B and C compared with group A(P<0.05).(4)With hematoxylin and eosin(H&E)staining,at 4 weeks after the cessation of distraction,abundant hypertrophic chondrocytes was visible in the center of distraction gap in group A,while the center of the distraction gap exists a small amount of fibrous tissue and cartilage tissue in group B and group C and the woven bone formed larger than that in group A.The distraction gap was filled with newly woven bone in group D.At 8 weeks after the cessation of distraction,the distraction gap of group A has been basically formed bone connection.But the number of bone trabecular was less,and its arrangement was irregular.The number and volume of bone trabecular in group B and C increased significantly compared with group A.The bone trabecular was much arranged orderly in group B compared with group C.The cortical bone basically formed in group D,and the medullary cavity was recanalized partly.The maturity of the new bone tissue was significantly better than the other three groups.(5)Biomechanical observed The femoral bending strength in A?C?B?D group increased gradually,there were differences between any two groups,(P<0.05.Conclusion Local application of SDF-1 or VEGF can promote the formation of new bone in the process of DO and both of them can shorten the period of treatment,but SDF-1 has stronger effect;SDF-1 combined application with VEGF can achieve the synergistic effects,and the effect of combined application is better than that of single factor application.The combination of these two growth factors provides a new and feasible method to shorten the period of treatment of DO. |
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