The Role Of CGRP In The Healing Process Of Mandible Fracture: A Experimental Study

Because bone tissue has a rich nerve supply, it has led to speculation that nerve fibers actively participate in bone metabolism. Clinical observations have indicated that bone growth, repair, and remodeling would be partly regulated by the local innervations. The calcitonin gene-related peptide (CGRP) is composed of 37 amino acids that are transcribed from the calcitonin (CT) gene by alternative splicing. CGRP is distributed throughout the central and peripheral nervous systems. In the peripheral nervous system, CGRP is found in both sensory and motor neurons and possesses multiple functions, such as neurotransmitter, vasodilator, and neurotrophic effector. Notwithstanding this, CGRP also takes part in the local regulation of bone growth and metabolism. Osteoprotegerin (OPG) is a novel secretory member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulated osteoclastogenesis. The receptor activator of the NF- κ B ligand(RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG, the ratio of OPG/RANKL in bone microenvironment is a key of osteoclast formation and differentiation. There were few researches about the relationship between CGRP and OPG/RANKL, so we performed in vivo and in vitro experiments to explore the relationship between CGRP and OPG/RANKL and the role of CGRP in the healing process of mandible fracture.Methods:Animal experiment: forty Rabbit was randomly divided into inferior alveolar nerve (IAN) transection plus mandible fracture group (n=20) (experimental group) and mandible fracture group (n=20) (control group). the fractured bone tissues were obtained and paraffin slices were made on the day7 , 14, 21, 28 after surgery respectively.Callus were subjected to HE and CGRP, OPG, RANKL immunohistochemical staining. The relationship of expression of OPG, RANKL and CGRP in Callus tissue among both denervation mandible fracture group(n=20)and mandible fracture group(n=20) was analyzed using SPSS10.0 system for windows.Cell culture:The osteoblasts isolated from newborm rabbit calvaria were cultured in media containinglO"10~10"7mol/Lrecombinant CGRP to observe the influence of CGRP on the expression of OPG and RANKL. Control groups without CGRP. After 24 hours of incubation, the expression of OPG and RANKL were observed through immunocytochemical methods and image analysis, semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR) was performed to detect the changes of OPG and RANKL mRNA expression ,with P -actin mRNA as control.Results:Animal experiment results: Result show that there are few CGRP immunoreactive nerve fibers in callus at 7 after IAN transection mandible fracture while a quantity CGRP immunoreactive nerve fibers in callus throughout the repair process in control group which has shown that experimental model of both groups are successful. The expression of OPG and RANKL in osteoblasts osteoclasts and fibroblast in both two groups . Compared with expression of RANKL and OPG protein in mandible fracture group, lower levels of OPG expression were detected throughout the repair process in denervation fracture groups while RANKL was strongly induced. The result of image analysis showed that there was a significant difference between both experimental groups and control groups. In addition, OPG protein expression changes in parallel with the changes of CGRP protein.in both groups, the expression of OPG protein reached peak level at 7 days after operation while RANKL expression is low. At 14 days, OPG expression began to decrease while RANKL expression remarkable increase.Cell culture results: Results showed that the Secondary osteoblasts were positively stained for Alkaline phosphatase (ALP), and cells mainly showed polygon and triangle. It proved that the cultured cells were rabbit osteoblast. Immunocytochemistry results showed that CGRP up-regulated the expression of OPG with dose-dependent manner, with the maximal effect at the concentration of 10'8~10-7 mol/L CGPR also down-regulated expression of RANKL with dose-dependent manner . With the maximal effect at the concentration of 10'7 mol/L CGPR. RT-PCR results showed that CGRP increased the expression level of OPG mRNA with a dose-depended manner while down-regulated RANKL mRNA expression with the same way.Conclusion: (1) hi mandible fracture process,the distribution of CGRP is concernedwith mandibular repair competence.it is indicated that CGRP exert important effection throughout mandible healing process. (2) The expression level of CGRP in callus can affect the volume and quality of bone callus directly. IAN transection contribute to deplete of CGRP ecpression which lead to defect of callus microstructure. (3) OPG/RANKL singal system is a juncture of information transfer between OB and OC. (4) The expression level of CGRP was positively correlated with expression of OPG, and negatively correlated with expression of RANKL.CGRP may up-regulate the ratio of expression of OPG / RANKL of osteoblast.it suggest that OPG/RANKL is mainly signal pathway of CGRP in the healing process of mandible fracture. (5) CGRP also regulates local blood supply, which play a part in mandible healing more or less.