| Objective: Calcitonin gene-related peptide(CGRP),as a neuropeptide substance,plays an important regulatory role in fracture healing.The receptor activator of nuclear factor κB(RANK)/receptor activator of NK-κB ligand(RANKL)/osteoprotegerin(OPG)system is also important for fracture healing.It was found in clinical practice that patients with spinal cord injury combined with fracture had faster callus formation,larger callus volume but poor biomechanical strength,which was a kind of pathological callus.In this research,the relationship between CGRP,RANK/RANKL/OPG system and pathological callus formation under denervalization was studied.Methods:In this research,96 healthy adult female SD rats aged 8-10 weeks were randomly divided into denervated + tibial fracture +CGRP group(group A),denervated + tibial fracture + normal saline group(group B),and simple tibial fracture + normal saline group(group C),with 32 rats in each group.Rats in group A and group B were treated with T10 spinal cord severed and tibia fracture model.Group C established tibial fracture model.Group A was treated with intraperitoneal injection of 1ml CGRP solution at the concentration of 5 μg/ml.Group B and C was treated with intraperitoneal injection of equal dose of normal saline.Tibial radiographs were taken on the 7th,14 th,21st and 28 th,and bone mineral density was detected on the 28 th.Tibial specimens were routinely decalcified,embedded and made into sections.8 tibial specimens were taken from each group at each time point.Sections were observed by HE staining and Masson staining.CGRP,RANKL and OPG were detected by immunohistochemical staining and analyzed statistically.Results:Imaging examination showed that callus formation in group A and B was faster and larger than group C,but the trabeculae showed loose and disorderly arrangement and the callus density was not uniform.Callus morphology in group A was better than group B.HE staining showed that the formation of fibrous callus and cartilage callus in group A and group B was faster than group C,but the arrangement of chondrocytes was relatively loose at 7 and 14 days.At 21 days,the cartilage callus was gradually replaced by new bone trabeculae.The trabeculae was loose in arrangement and its number was small in group B.Group A were slightly better than group B.In group C,a large number of trabeculae were formed and closely arranged.At 28 days,fracture healing was good in group C.Basic fracture healing was finished in group A with relatively loose trabecular alignment.The situation of group B was worse than group A and a small amount of fibrous callus covering the lateral edge of new bone.Masson staining showed that at each time point,the percentage of mature bone in group B was lower than group A,and group A was lower than group C,especially at 14,21 and 28 days,the percentage of mature bone in each group was statistically significantly different(P<0.05).Immunohistochemical staining showed that CGRP expression was high level in group C,medium level in group A,low level in group B,with statistically significant difference(P<0.05)at 14 and 21 days.At 14 days,RANKL expression was high level in group B,medium level in group A,low level in group C which was statistically significant(P<0.05).The expression level of RANKL in group C was still lower than group A and group B,which was statistically significant(P<0.05).At 7 days,OPG expression was medium level in group A,low level in group B,and high level in group C.The difference in OPG expression between group B and group C was statistically significant(P<0.05).At 14 days,OPG expression of group C was higher than group A and group A was higher than group B,with statistically significant differences(P<0.05).At 21 days,the OPG expression of group C was higher than group A and group B,with statistically significant difference(P<0.05).At 28 days,OPG expression was increased in the three groups,and the difference between group B and group C was statistically significant(P<0.05).Conclusion: CGRP and RANK/RANKL/OPG system play important roles in the process of fracture healing,and CGRP has certain regulatory capacity to the RANK/RANKL/OPG system.A large number of pathological callus were formed during denervated fracture healing,which may be related to the low expression of CGRP around the fracture and the weakened ability of CGRP to regulate RANK/RANKL/OPG system. |
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