神经生长因子降钙素基因相关肽在下颌骨骨缺损愈合时调节机制研究
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摘要
因外伤、肿瘤、先天性畸形等造成颌骨缺损的修复一直是困扰口腔颌面外科临床医生的难题。颌骨缺损一方面造成功能障碍，另一方面造成美观问题，加重患者的心理负担。随着社会的进步和发展，交通事故的发生机会也越来越多，头面部损伤和颌骨骨折的发生率呈增高趋势。现代战争中，面部缺乏防护，颌面部现代战伤发生率超出10%。因此，有关颌骨骨缺损修复的研究也一直是基础和临床研究的热点问题。
骨缺损的修复中，神经系统在其中发挥着重要的调节作用。我们的前期研究发现，神经介质在修复的启动、成骨和后期改建中均发挥重要作用，有许多细胞因子参与这一过程。分子生物学研究表明，骨折愈合是一个多种细胞生长因子参与调控的复杂过程，这其中既有骨缺损周围成骨相关细胞分泌的细胞因子作用，也有远端相关器官通过体液转运而来的各种激素和调节因子，还有神经系统通过神经末梢释放出的神经递质参与其中。神经生长因子(nerve growth factor，NGF)是神经营养因子(neurotrophicfactor； neurotrophic factors，NTF)的一种，也是骨缺损周围成骨相关细胞分泌的一种细胞因子。研究表明，NGF对骨缺损愈合过程起着重要的调节作用。降钙素基因相关肽(calcitonin gene-related peptide，CGRP)作为重要的感觉神经信号分子，因在骨折愈合调控中的重要作用而受到广泛关注。NGF与CGRP都与骨缺损愈合过程相关，并且国内外诸多研究发现CGRP和NGF骨折区的含量均增多。而且都与支配骨组织的神经系统有关联，不同在于NGF是神经靶细胞分泌，被神经末梢摄取的，CGRP是神经末梢释放的，作用于靶细胞的。这两者在骨愈合过程中的相互关系如何？有没有相互之间的调节机制？
基于以上想法，并结合国内外研究进展，本课题首先制备可以在骨缺损区域持续释放NGF的缓释微球，研究下颌骨骨缺损愈合过程中NGF对支配下颌骨的三叉神经节中CGRP的合成与分泌的影响。利用药剂学技术，制备单甲氧基聚乙二醇聚乳酸神经生长因子[(mono-methoxy poly (ethylene glycol)-poly(lactic acid)-nerve growth factor，mPEG-PLA-NGF]缓释微球，检测微球的包封率、载药量等理化性质，研究缓释微球国家自然科学基金资助项目(编号30973334）；重庆市自然科学基金（CSTC2009BB5334）的体外释放规律。利用PC-12细胞培养实验，检测微球释放NGF的生物活性和临床实际应用价值。制备大鼠下颌骨骨缺损模型，通过全身注射或局部应用NGF缓释微球干预骨愈合过程，检测大鼠三叉神经节内NGF、CGRP、TrkA(tyrosine kinase A，络氨酸激酶A)等的变化，探讨下颌骨骨缺损愈合过程中NGF对神经节的作用。原代培养大鼠的成骨细胞，并进行形态观察和鉴定，用不同浓度的CGRP刺激成骨细胞，观察CGRP对成骨细胞合成NGF的影响
实验方法和结果：
1、 mPEG-PLA-NGF缓释微球的制备及体外释放实验研究
mPEG-PLA-BSA微球的制备预实验：取三种不同配比的单甲氧基聚乙二醇聚乳酸(mono-methoxy poly (ethylene glycol)-poly(lactic acid)，mPEG-PLA)(mPEG：PLA分别为1:4，1:10，1:20的3种mPEG-PLA，其中mPEG分子量均为5000。)用复乳法制备微球，通过筛选，选用mPEG：PLA=1:20的mPEG-PLA。用复乳法制备mPEG-PLA-NGF缓释微球，观察微球的表面形态，微球表面光滑圆整，球体大小较均匀。微球平均粒径为74.2±21.3μm，粒径分布范围较窄。载药量和包封率的实验表明，mPEG-PLA-NGF缓释微球包封率为(77.3±1.81)％。载药量为[(2.13±0.24)×10-5]%。微球的体外释放实验表明，微球在14d内，不仅一直持续释放神经生长因子，而且所释放出的神经生长因子浓度可以保持在一定的水平：(54.67-76.81) fmol/μL，平均浓度为(65.74±11.07) fmol/μL。经检测表明，神经生长因子缓释微球的24h内释放量为27.36％，21d后释放度达72.34％。NGF缓释系统中释放的NGF活性测定试验中，通过PC-12细胞的出芽情况证明了mPEG-PLA-NGF缓释微球可以持续释放出有生理活性的NGF。
2、神经生长因子在下颌骨骨缺损愈合过程中对骨折愈合的促进作用以及对三叉神经节合成降钙素基因相关肽的影响
建立大鼠下颌骨骨缺损动物模型，分为：①空白组：只手术，不给药；②对照组：手术后，NGF腹腔给药；③实验组：手术时，局部涂布mPEG-PLA-NGF缓释微球。分别在术后1w，2w，4w，8w处死动物。术区标本HE染色显示，mPEG-PLA-NGF实验组的骨痂生成量和骨痂成熟度要明显高于对照组和空白组，而且愈合时间明显提前。四环素荧光染色结果显示，mPEG-PLA-NGF实验组可加快大鼠新生骨的形成，结果有统计学意义。
建立大鼠下颌骨骨缺损动物模型，分为：①单纯骨缺损组，不给药；②单纯全身给药组，不手术；③骨缺损+全身给药组；④骨缺损+NGF微球；⑤空白组。在术后第1d，2d，3d，7d处死动物，获取三叉神经节标本，并通过免疫组化法，检测三叉神经节中NGF、CGRP、TrkA的表达差异。结果显示，各组均观察到三叉神经节内NGF、CGRP、TrkA表达有所增强，第2组最不明显，第4组表达显著高于其他组。3天达到最高，以后逐渐下降，但仍高于两对照组。三项指标结果极为相似，显示出密切的相关性。
3、降钙素基因相关肽对成骨细胞分泌神经生长因子的影响
原代培养大鼠成骨细胞，并进行了形态观察和鉴定。用不同浓度的CGRP刺激成骨细胞，在第1，2，3，5d，用ELISA法测不同浓度CGRP对大鼠成骨细胞上清液中NGF浓度的影响。结果显示成骨细胞自身可以产生少量的NGF，在CGRP作用下，可以明显上调成骨细胞分泌的NGF，而且随CGRP浓度升高，此作用也相应增强。此外，成骨细胞上清液中NGF的浓度随CGRP作用后时间延长也增加。
结论：
1、以mPEG-PLA为载体，采用复乳法(w/o/w)制备的mPEG-PLA-NGF缓释微球具有良好的物理和生化性能，能够持续释放具有生物活性的神经生长因子。
2、实验采用的微球制备方法和工艺确实可行，并具有一定的实用性。通过中国、美国、欧盟、日本等专利检索，“一种生长因子缓释微球及其制备方法”具有原创性，已经获得了中国知识产权局技术发明专利(专利号： ZL201110027337.8)。
3、体内实验表明骨缺损区域的NGF能够促进大鼠下颌骨骨缺损的愈合，促进大鼠的三叉神经节细胞合成CGRP。
4、体外研究表明CGRP对成骨细胞NGF的表达有促进作用，并与剂量和时间呈正相关。
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 vitro
Preliminary 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 osteoblast
The 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.
骨缺损的修复中，神经系统在其中发挥着重要的调节作用。我们的前期研究发现，神经介质在修复的启动、成骨和后期改建中均发挥重要作用，有许多细胞因子参与这一过程。分子生物学研究表明，骨折愈合是一个多种细胞生长因子参与调控的复杂过程，这其中既有骨缺损周围成骨相关细胞分泌的细胞因子作用，也有远端相关器官通过体液转运而来的各种激素和调节因子，还有神经系统通过神经末梢释放出的神经递质参与其中。神经生长因子(nerve growth factor，NGF)是神经营养因子(neurotrophicfactor； neurotrophic factors，NTF)的一种，也是骨缺损周围成骨相关细胞分泌的一种细胞因子。研究表明，NGF对骨缺损愈合过程起着重要的调节作用。降钙素基因相关肽(calcitonin gene-related peptide，CGRP)作为重要的感觉神经信号分子，因在骨折愈合调控中的重要作用而受到广泛关注。NGF与CGRP都与骨缺损愈合过程相关，并且国内外诸多研究发现CGRP和NGF骨折区的含量均增多。而且都与支配骨组织的神经系统有关联，不同在于NGF是神经靶细胞分泌，被神经末梢摄取的，CGRP是神经末梢释放的，作用于靶细胞的。这两者在骨愈合过程中的相互关系如何？有没有相互之间的调节机制？
基于以上想法，并结合国内外研究进展，本课题首先制备可以在骨缺损区域持续释放NGF的缓释微球，研究下颌骨骨缺损愈合过程中NGF对支配下颌骨的三叉神经节中CGRP的合成与分泌的影响。利用药剂学技术，制备单甲氧基聚乙二醇聚乳酸神经生长因子[(mono-methoxy poly (ethylene glycol)-poly(lactic acid)-nerve growth factor，mPEG-PLA-NGF]缓释微球，检测微球的包封率、载药量等理化性质，研究缓释微球国家自然科学基金资助项目(编号30973334）；重庆市自然科学基金（CSTC2009BB5334）的体外释放规律。利用PC-12细胞培养实验，检测微球释放NGF的生物活性和临床实际应用价值。制备大鼠下颌骨骨缺损模型，通过全身注射或局部应用NGF缓释微球干预骨愈合过程，检测大鼠三叉神经节内NGF、CGRP、TrkA(tyrosine kinase A，络氨酸激酶A)等的变化，探讨下颌骨骨缺损愈合过程中NGF对神经节的作用。原代培养大鼠的成骨细胞，并进行形态观察和鉴定，用不同浓度的CGRP刺激成骨细胞，观察CGRP对成骨细胞合成NGF的影响
实验方法和结果：
1、 mPEG-PLA-NGF缓释微球的制备及体外释放实验研究
mPEG-PLA-BSA微球的制备预实验：取三种不同配比的单甲氧基聚乙二醇聚乳酸(mono-methoxy poly (ethylene glycol)-poly(lactic acid)，mPEG-PLA)(mPEG：PLA分别为1:4，1:10，1:20的3种mPEG-PLA，其中mPEG分子量均为5000。)用复乳法制备微球，通过筛选，选用mPEG：PLA=1:20的mPEG-PLA。用复乳法制备mPEG-PLA-NGF缓释微球，观察微球的表面形态，微球表面光滑圆整，球体大小较均匀。微球平均粒径为74.2±21.3μm，粒径分布范围较窄。载药量和包封率的实验表明，mPEG-PLA-NGF缓释微球包封率为(77.3±1.81)％。载药量为[(2.13±0.24)×10-5]%。微球的体外释放实验表明，微球在14d内，不仅一直持续释放神经生长因子，而且所释放出的神经生长因子浓度可以保持在一定的水平：(54.67-76.81) fmol/μL，平均浓度为(65.74±11.07) fmol/μL。经检测表明，神经生长因子缓释微球的24h内释放量为27.36％，21d后释放度达72.34％。NGF缓释系统中释放的NGF活性测定试验中，通过PC-12细胞的出芽情况证明了mPEG-PLA-NGF缓释微球可以持续释放出有生理活性的NGF。
2、神经生长因子在下颌骨骨缺损愈合过程中对骨折愈合的促进作用以及对三叉神经节合成降钙素基因相关肽的影响
建立大鼠下颌骨骨缺损动物模型，分为：①空白组：只手术，不给药；②对照组：手术后，NGF腹腔给药；③实验组：手术时，局部涂布mPEG-PLA-NGF缓释微球。分别在术后1w，2w，4w，8w处死动物。术区标本HE染色显示，mPEG-PLA-NGF实验组的骨痂生成量和骨痂成熟度要明显高于对照组和空白组，而且愈合时间明显提前。四环素荧光染色结果显示，mPEG-PLA-NGF实验组可加快大鼠新生骨的形成，结果有统计学意义。
建立大鼠下颌骨骨缺损动物模型，分为：①单纯骨缺损组，不给药；②单纯全身给药组，不手术；③骨缺损+全身给药组；④骨缺损+NGF微球；⑤空白组。在术后第1d，2d，3d，7d处死动物，获取三叉神经节标本，并通过免疫组化法，检测三叉神经节中NGF、CGRP、TrkA的表达差异。结果显示，各组均观察到三叉神经节内NGF、CGRP、TrkA表达有所增强，第2组最不明显，第4组表达显著高于其他组。3天达到最高，以后逐渐下降，但仍高于两对照组。三项指标结果极为相似，显示出密切的相关性。
3、降钙素基因相关肽对成骨细胞分泌神经生长因子的影响
原代培养大鼠成骨细胞，并进行了形态观察和鉴定。用不同浓度的CGRP刺激成骨细胞，在第1，2，3，5d，用ELISA法测不同浓度CGRP对大鼠成骨细胞上清液中NGF浓度的影响。结果显示成骨细胞自身可以产生少量的NGF，在CGRP作用下，可以明显上调成骨细胞分泌的NGF，而且随CGRP浓度升高，此作用也相应增强。此外，成骨细胞上清液中NGF的浓度随CGRP作用后时间延长也增加。
结论：
1、以mPEG-PLA为载体，采用复乳法(w/o/w)制备的mPEG-PLA-NGF缓释微球具有良好的物理和生化性能，能够持续释放具有生物活性的神经生长因子。
2、实验采用的微球制备方法和工艺确实可行，并具有一定的实用性。通过中国、美国、欧盟、日本等专利检索，“一种生长因子缓释微球及其制备方法”具有原创性，已经获得了中国知识产权局技术发明专利(专利号： ZL201110027337.8)。
3、体内实验表明骨缺损区域的NGF能够促进大鼠下颌骨骨缺损的愈合，促进大鼠的三叉神经节细胞合成CGRP。
4、体外研究表明CGRP对成骨细胞NGF的表达有促进作用，并与剂量和时间呈正相关。
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 vitro
Preliminary 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 osteoblast
The 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.
引文
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