An Experiment Study On The Mechanism Of Nerve Growth Factor And Calcitonin Gene Related Peptide Promoting Mandible Defect Repair

There is a terrible difficulty in mandible defect repair after injury for clinical doctorsdue to trauma, tumor, congenital malformation and so on. On one hand, jaw defect inducesdysfunction, and aggravates the patient burden because of preventing beautiful outlook onanother hand. During the social development, the ratio of traffic accidents step up more andmore meanwhile the ratio of jaw fractures happen as well; and the ratio of jaw fractures inthe war is critical high as well as above, thus, it makes the research of the restoration afterjaw fractures to be a hot spot. The restoration after jaw injury is based on the stability andblood supply surrounding the jaw defect, there are many cytokines participated in it, andthere is the important role for nerve growth factor in regulation function.The molecular biological research showed, the jaw restitution was is a complexprocess in which many cytokines took place, and there were many cytokines producingmarked effect, included cytokines with cells secreting related to osteogenesis surroundingjaw defect, kinds of hormones and regulatory factors transported from distal organ by bodyfluids and neurotransmitters released from nerve terminal in nerve system. Nerve growthfactor is one of the nerve nutrition factors, is also secreted from the cells related toosteogenesis surrounding bone defect. The research showed, NGF played an important rolein the process of restoration after jaw defect. As the considerable sensory nerve signalingmolecule, calcitonin gene related protein is paid close attention very much due to itsimportant function in regulation of restoration of jaw fractures. NGF and CGRP are allrelated to the restoration after bone fractures, and the internal and abroad research foundthat CGRP and NGF released from the position of fractures increased apparently, Moreoverthey were all related to nerve system gaverning bone tissue, but the differences betweenthem were that NGF was secreted from nerve target cells and was uptake by nerve terminal,CGRP was released from nerve terminal and acted at target cells. Is there some relationship or how to interact between each other in process of bone restitution?Based on above information, and binding internal and abroad research, we firstproduced the microballoons which could released NGF continuously and slowly at theposition of defect, order that investigate the effect of NGF which governed mandibletrigeminal nerve in the restoration after mandible fractures, specially influence on synthesisand secretion of CGRP. We used pharmaceutics technology to produce mPEG-PLA-NGFmicroballoons, detected physical and chemical characteristic of microballoons, included theentrapment rate, drug-loading rate and so on; investigated the release pattern ofmicroballoons in vitro; used PC-12cell culture experiment to detect if there was biologicalactivity in NGF released from microballoons. All of above were to do the groundwork fornext experiments. Meanwhile, there was some practical application value. We prepared themodel of mandible defect of rat, through NGF released from microballoons which injectedlocally and systemic administration in the process of bone restitution, detected the changesof NGF, CGRP, TrkA in ganglia of rat trigeminal nerve, and investigated the influence ofNGF on ganglia in restoration after mandible defects. We cultured primarily, observedmorphologically and identified the rat osteoblast, then stimulated osteoblast with differentdensity of CGRP to observe the influence of CGRP on synthesis of NGF of osteoblast.Method and Result:1. the produce of mPEG-PLA-NGF microballoons and release experiment in vitroPreliminary experiment of the the produce of mPEG-PLA-NGF microballoons: usedthree kinds of matches mPEG-PLA(mPEG: PLA=1:4,1:10,1:20, and Molecular Weight ofmPEG is5000) to produce microballoons by multiple emulsion, then identified, selectedmPEG:PLA=1:20. We produced mPEG-PLA-NGF microballoons by multiple emulsion,and observed surface appearance of microballoons, results showed the surface ofmicroballoons was smooth and clean, microballoons were well-distributed. The average ofparticle diameter of microballoons was74.2±21.3μm, distribution range of particlediameter was a little narrow. The results of drug-loading rate and entrapment rateexperiments indicated that microballoons do not only release continuously nerve growthfactors, but also make the density of NGF keep a stable level during14days:(54.67-76.81)fmol/μL, the average density was (65.74±11.07) fmol/μL. Detection showed the volume ofNGF released from microballoons were27.36％in24h,72.34％in21d. In the detection experiment of activity of NGF released from release system, it proves thatmPEG-PLA-NGF microballoons could release continuously NGF with activity byprolification of PC-12cell.2. The promotion of NGF for fracture healing during the mandible restitution afterbone defect and the effect of NGF for CGRP synthesized by trigeminal ganglion.We constructed the model of rat mandible bone defect, include:①blank group: justoperation, no medicine;②control group: after operation, NGF of Intraperitonealadministration;③experimental group: mPEG-PLA-NGF microballoons was coatedpartially in operation. Then the animals were sacrificed at1w,2w,4w,8w after operation.HE results showed: the quantity and maturation of mPEG-PLA-NGF experimental groupwere significantly better than control group and blank group, and the healing time wasapparently advanced. Tetracycline staining results showed, the new bone formation of ratwas promoted in mPEG-PLA-NGF experimental group, significantly.We constructed the model of rat mandible bone defects, included:①simple-bonedefects group, with no medicine;②simple systemic administration, no operation;③bonedefects+systemic administration group;④bone defects+NGF microballoons;⑤blankcontrol group. The animals were sacrificed at first, second, third, seventh day afteroperation, ganglia of trigeminal nerve was gained, and the difference of expression of NGF,CGRP, TrkA in ganglia were detected by immunohistochemisty. Findings showed:expression of NGF, CGRP, TrkA in ganglia were increased in each group, the expression ofsecond group was lowest, the expression of forth group was apparently higher than others,the highest at the third day, then downregulated gradually, but still higher than two controlgroups. The results of these three markers were so similar that there were close associationbetween them.3. The influence of CGRP on NGF synthesized by osteoblastThe rat osteoblast was primary cultured, observed morphologically and identified. Theosteoblast was stimulated by CGRP with different density, at1,2,3,5d, the influence ofdifferent density CGRP on density of NGF in the supernatant of rat osteoblast by ELISA.Results showed osteoblast can release a little NGF, and can release more NGF under theCGRP working; moreover, the effectiveness of CGRP was stronger during density of CGRPupregulation. Conclusion:1. There were very well physical and chemical characteristics in mPEG-PLA-NGFmicroballoons with mPGE-PLA carrier by double emulsion, which could releasecontinuously NGF with activity.2. the method and technology of producing microballoons was real working, and it hassome practicality.“a kind of NGF released from microballoons and producing technology”was original by patent search from china, USA, UE, Japan and so on, and it had beenacquired China Intellectual Property Office technical invention patent(No.201110027337.8).3. Results indicated that NGF could accelerate the process of mandible defect repairand promote synthesis of CGRP in ganglion cells of trigeminal nerve at the position of bonedefects in vivo.4. Results indicated that CGRP could promote the expression of NGF in osteoblast invitro, and be positively correlated with the dose and the event.