The Effects Of CGRP On The Expression And Activity Of TGF-β1in The Healing Of Mandibular Fracture In Rabbits

Calcatonin gene related peptide （CGRP）, as a peptide neurotransmitters is mainlydistributed in bone tissue which the metabolism is active.It can regulate bone cell’s activity,growth, differentiation, thus regulate the progress of fracture healing. But the specificregulation mechanism of CGRP in bone is still not clear. Study found that transformatinggrowth factor-β （TGF-beta） can stimulate and promote the mesenchymal stem cells（MSC）,cartilage cells and osteoblasts proliferation, and can also induce extracellular proteinssynthesis.As a result,it play an important role in the formation of cartilage and boneformation in rebuilding period. At present five different structure of the TGF-β havealready been found. Different sub-structure TGF-β has reaches the express peak in thedifferent progress of fracture healing and their regulation effects are also different. TGF-β₁and TGF-β2’s ratio is about4：1and TGF-β₁expression time is the longest.It is not clearabout CGRP could regulate the metabolism of TGF-β₁, thus promote the fracture healingprogress.This experiment is to establish the mandible fracture model under lost alveolar nerve.The distribution of CGRP in callus was detected, and then studies the effects of CGRP’sexpression in jaw bone fracture healing callus on the expression of TGF-β₁. Osteoblast wascultivated in vitro, exogenous CGRP were added in osteoblast, then the expression ofTGF-β₁in different time were detected. Thus to explore the relation about CGRP andTGF-β₁in progress in bone fracture healing.Methods：Cell experiment: To observe the influence of CGRP on TGF-β₁expression, new rabbitskull osteoblasts were cultured and treated with exogenous CGRP, then expression ofTGF-β₁and cell viabilities were also assessed. Animal experiment: This experiment is to establish animal models of lost alveolarnerve jaw bone fractures and unsophisticated fracture healing groups （25for each）. Twogroups of animals were killed via air embolism death in the ear edge in4,7,14,21,28days,respectively. The original callus of the fracture was obtained. Using theimmunohistochemistry, the healing speed and quality of the callus in two groups of animalswere assessed throught TGF-β₁protein expression detected by western blot method.Results:Cell experimental results:（1） Osteoblasts of rabbit were successfully separated andcultivated and identified using dyeing alkaline phosphatase （ALP）.（2） The cell viabilitieswere significantly increased after treatment of exogenous CGRP.（3） The cell viabilitiesincreased with the increase of CGRP concentration. This tendency is more obvious among48h and72h group （P <0.05）.（4） TGF-β₁protein expression increased significantly withthe increase of the concentration of CGRP （P <0.05）. Hence, CGRP may promote theTGF-β₁expression of rabbit osteoblasts.Animal experiment results:（1） Under high magnification microscopy, new bone growsquickly in simple fracture group callus tissue. The arrangement of the trabecular meshworkrules is regular and bulky density, whereas lost alveolar nerve bone trabeculae is sparse. Itsarrangement is irregular, and callus structure is defected.（2） The proliferating smallcartilage osteoblast was TGF-β₁positive14days after treatment of CCRP assess byimmunohistochemical staining. The calcification converted to new bone cartilage and thesurface osteoblasts, with TGF-β₁dyeing strong positive.（3） Western blot analysis showedthat TGF-β₁protein expression gradually increased, and reached its highest expression14days after management. Then TGF-β₁protein expression kept stabilized. At the14th dayafter management, TGF-β₁protein expression was significantly lower in Lost nerve groupthan those in the simple nerve fracture group.Conclusion:（1）This experiment successfully constructed lost alveolar nerve animalmodels, which effectively reduce CGRP distribution and expression in fracture healingparts,leading to obvious defects and callus repairing at a slow rate.（2） CGRP has astimulative effect on the osteoblast proliferation and mineralization ability.（3） In the jawbone fracture healing process, CGRP nerve fiber volume and the release of bone repairability closely related to CGRP, indicating that CGRP plays an important role in the reconstruction of the fracture.（4） In the process of callus growth，TGF-β₁expression hasobvious timeliness, playing a critical role during fracture healing in the different stages anddirectly control the fracture healing process.（5） Cell experiment and animal experimentshave shown that CGRP expression and TGF-β₁expression are positively correlated, andCGRP thereby may raise cell TGF-β₁expression.