| The nervous system involves in the regulation of the process of repair and remodeling of fracture and bone metabolism at different levels. The mechanism, of which systemic and local factors cooperate with nervous system, is quite complicated. However, the regulation achieves finally through the effect of neural signals on function of osteoblasts and osteoclasts in bone, which are released from the nerve terminals distributed in bone[1-5]. Calcitonin gene-related peptide (CGRP) secrected by sensory nerve has many kinds of physiological activities. Previous studies have found that CGRP is involved in repair and remodeling of fracture through influencing functions of osteoblasts, and little is known about the molecular mechanism and cell sigal transduction of CGRP's modulating the healing process of fracture at the present time.In our previous studies, we explored the effect of inferior alveolar nerve(IAN) amputation on the healing of mandibular fracture and its possible mechanism at the level of tissue[6,7]. To explore the molecular mechanism of the nervous system's modulating fracture healing, in this study we will explore further the effect of CGRP on mandibular fracture healing at the level of cell with bone histomorphometry through the foundation of animal model of mandibular fracture with or without IAN amputation, and clarify cell signal transduction of osteoblasts at the level of molecule by the primary rabbit osteoblast cultures of neonatal calvaria.PurposeThe aims of the study were to explore the possible mechanism of CGRP's modulating the healing of rabbit mandibular fracture and cell signal transduction of rabbit cultured osteoblast acted by hCGRP, and so that to explore further the molecular mechanism of sensory nerve's modulating fracture healing.Methods105 adult China white rabbits were randomly divided into two groups (experimental and control groups). They all suffered from a fracture in the left mandible. Animals in experimental group were injected by LY303870, an antagonist to Substance P receptor, without IAN amputation. The control group was divided into two groups, and half of them with or without IAN amputation. The rabbits were sacrified on 7, 14, 21 and 28 day after operation, and the specimens were collected and stained with hematoxylin-eosin and for immunochemistry to observe the process of fracture healing and the expression of SP and CGRP in bone calllus, and stained with Goldner's Masson Trichrome stain and the double marks of tetracycline to analyse the newly callus quantitively with bone histomorphometry. Osteoblast cultures were isolated from newly-born rabbit calvaria by sequential enzymatic digestion. The cultured osteoblasts were divided into six groups according to the final concentration of CGRP. The control group was divided into two groups. One is DMEM without CGRP or CGRP(8-37), and the other is DMEM with 10-9mol/L CGRP and 10-6mol/L CGRP(8-37) together. The other four groups are DMEM with 10-10mol/L, 10-9mol/L, 10-8mol/L and 10-7mol/L CGRP respectively and without CGRP(8-37). The detection of cAMP accumulation in cultured cells was performed using radioimmunology assay and the change of cyto free Ca2+ concentration was monitored by laser scanning confocal microscopy. The activity of activiting transcription factor 4(ATF4) was measured in electrophoretic mobility shift assay, and the expression and their mRNA of ATF4, osteocalcin, receptor activator of nulear factor-κB ligand and osteoprotegin in cultured cells were detected by Western Blot and Northern Blot respectively.Results:The expression of SP and CGRP in cullus:Immunoreactivities of SP in callus occurred strong in the group without IAN amputation on Day 7 after operation, and became stronger on Day 14 and less on Day 21 after operation, and became stronger again on Day 28 after operation. Immunoreactivities of CGRP in callus occurred strong in the group without IAN amptation, became the strongest on Day 14 and less stronger gradually on Day 21 and 28 after operation. Immonoreactivities of SP and CGRP in callus occurred weakly at early stage and became stronger at late one in the group with IAN amputation. There were significant differences in the expression of SP and CGRP in callus between two groups.The effect of CGRP on the healing of mandibular fracture: there were no significant differences in ob?s/BS and ob?Nb/BPm among three groups. On the contrary, there were significant differences among three groups in oc?s.BS, N?oc/BS, BFR, OS and MLV by analysing the callus using bone morphometry.The effect of hCGRP on free Ca2+ concentration in rabbit cultured osteoblasts: hCGRP can't increase cyto free Ca2+ concentration in cultured osteoblasts. There were no signifficant differences among three groups.The effect of hCGRP on cAMP concentration in rabbit cultured osteoblasts: hCGRP can stimulate the accumulation of cAMP in cultured osteoblasts. And it displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).The effect of hCGRP on ATF4 activity in cultured osteoblasts: hCGRP can promot the activities of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can enhance the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can promote the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).The effect of hCGRP on the expression of OPG and its mRNA in cultured osteoblasts: hCGRP can promote the expression of OPG and up-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).The effect of hCGRP on the expression of RANKL and its mRNA in cultured osteoblasts: hCGRP can inhibit the expression of RANKL and down-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).All the obtained data in vitro experiments were evaluated by the statistical analysis using Pearson's correlation coefficient to describe the degree of corelation in each index. And there was significant difference in corelation among cAMP, ATF4, OC, OPG and RANKL. Conlusion:1. The injury of IAN can influence the quality of newly callus through affecting the mineralization and remodeling of bone callus.2. The expression of SP and CGRP in callus is dynamic, which is coherent with the healing process of fracture. It suggests that nervous system's modulating the healing process of fracture may achieve by neuropeptides releasing from the nerve ending distributed in bone.3. The involvement of CGRP in regulation of the healing process of fracture may achieve through affecting the function of osteoblasts and the function and differentiation of osteoclasts.4. It may be one of the cell signal transductions of rabbit cultured osteoblast that the hCGRP's binding to CRLR can activate AC and promote the accumulation of cAMP, as a result, PKA may be activated and stimulate phosphorylation and accumulation of ATF4, and at last induce osteoblast-specific gene expressioin.5. In order to modulate the repair and remodeling of fracture, CGRP may affect the production of OPG and RNAKL in osteoblasts and accordingly influence the differentiation of osteoclasts. |
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