力生长因子E肽在临界骨缺损修复中的作用及机制
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摘要
肿瘤、创伤、感染等因素所造成的大面积骨缺损修复一直是困扰骨科医生的难题。目前，骨缺损的治疗主要还是依赖传统的骨移植术，但传统方式一般会经历多次手术、存在诸多副作用及因手术失败引起的截肢或丧失骨负重等可能性，因此迫切需要新的替代方法以满足临床治疗需求。生长因子的应用为骨缺损治疗提供了一种可供选择的治疗方式。目前经FDA批准的用于临床骨修复的生长因子主要是BMPs家族蛋白，其中rhBMP-2的活性最强，但在临床运用中rhBMP-2却存在治疗费用较高，有效剂量难以控制易导致异位成骨、过量成骨、新生骨快速吸收等成骨异常现象出现。本文针对rhBMP-2在临床运用中出现的问题，试图寻找到一种替代因子或与rhBMP-2联用能解决其成骨异常问题的因子。力生长因子E肽(C-terminal24-amino acid peptide of mechano growth factor, MGF24E)是近年来引起广泛关注的一个功能活性多肽，是受损组织表达的一种IGF-I选择性剪接变体，实验已证明它能保护受损组织及细胞，促进损伤修复。基于本课题组对MGF24E的前期研究成果，本研究从影像学、组织学以及生物力学等多方面证实了力生长因子E肽(MGF24E)以及MGF24E+rhBMP-2联合用药在临界骨缺损修复中的积极作用，同时在细胞学水平、基因水平以及蛋白质水平探讨了损伤修复机制。研究的主要内容和所取得的结果如下：
1.在制备了1mm左右间隙的SD大鼠桡骨开放性骨折模型的基础上，利用前期制备的MGF/MGF24E兔抗人多抗，采用Western blot技术检测和分析大鼠骨折修复组织中MGF蛋白的表达情况。结果显示截骨损伤组织中MGF蛋白表达在术后1天即上升到较高值，术后第3天达到最高值，随后表达量虽有下降但仍维持较高表达。
2.从影像学和组织学的观察及分析角度，综合评价外源MGF24E注射修复兔桡骨节段性骨折的效果。
①高剂量MGF24E修复兔桡骨5mm节段性骨折的临床愈合时间，较MGF24E低剂量组和阴性对照组提早2周；MGF24E高剂量组重建骨组织为髓腔贯通的管状骨，其修复情况明显好于MGF24E低剂量组和阴性对照组；
②高剂量MGF24E能显著促进骨折重建组织的血管新生，组织学评分结果显示高剂量组样本新生血管数为阴性对照组的2倍以上。
3.采用2%FBS血清饥饿细胞模型，以PBS为阴性对照、100ng/mL VEGF165为阳性对照，考察MGF24E对人静脉内皮EA.hy926细胞增殖、迁移和体外管腔形成的影响；以PBS为阴性对照，10%FBS正常培养条件为阳性对照，考察10ng/mLMGF24E对人静脉内皮EA.hy926细胞VEGF和Ang-I的mRNA及蛋白表达的影响；同时还研究了MAPK/ERK通路在MGF24E影响血管内皮细胞体外血管化细胞行为及其分子机制中的作用。
①10ng/mL MGF24E促细胞增殖能力等效于100ng/mLVEGF165，MGF24E促细胞增殖是通过增加S期细胞数目和促进细胞DNA合成的方式完成，并完全依赖MAPK/ERK信号通路；
②MGF24E促细胞迁移、管腔形成的能力明显强于PBS阴性对照，但弱于VEGF165，并发现MAPK/ERK通路部分参与了MGF24E促细胞迁移、管腔形成等事件；
③MGF24E能逆转血清饥饿引起的正常生长细胞VEGF和Ang-I表达下调的趋势，其上调量高于正常培养组的表达量，并发现MAPK/ERK通路部分参与了MGF24E上调VEGF和Ang-I表达的整个过程。
4.采用7%FBS血清饥饿细胞模型，以PBS为阴性对照、100ng/mLVEGF165+50ng/mL rhBMP-2的联合用药为阳性对照，25ng/mL MGF24E、50ng/mLrhBMP-2单给药为联合用药组的增效参照，考察25ng/mL MGF24E+50ng/mLrhBMP-2联合用药对原代成骨细胞增殖、迁移、分化早期指标ALP活性以及矿化钙分泌量表达的影响，并研究其分子机制。
①MGF24E促细胞增殖能力强于rhBMP-2和PBS，相对于MGF24E或rhBMP-2单独给药，MGF24E+rhBMP-2联合用药对促进细胞DNA合成、迁移和分化矿化有协同增效作用；阳性对照除对促进细胞迁移有协同增效作用外，在其他成骨相关细胞生理行为上均表现为拮抗抑制作用；
②MGF24E+rhBMP-2联合用药在细胞分化矿化事件上的协同增效作用主要涉及调控成骨信号通路的转录因子Runx2基因和骨基质形成的OPN基因表达上调。
5.以可吸收明胶海绵(Absorbable Gelatin Sponge, AGS)作为载体，采用低温负压物理吸附法制备4种不同给药方案的复合生长因子支架，并对复合支架的形貌结构、孔隙率、生长因子的体外释放以及支架细胞相容性等性能进行评价。
①扫描电镜结果显示，生长因子成球状分布于复合AGS支架内部，支架孔径和孔隙率能满足成骨细胞长入；
②体外释放结果显示，不同复合支架中生长因子的体外释放均发生突释现象，其中MGF24E释放最快，rhBMP-2释放最慢，生长因子的释放规律可满足机体对MGF24E需求以及临床上rhBMP-2的一次性高浓度给药方式；
③复合支架中成骨细胞活性实验结果显示，各组复合支架表现出细胞相容性差异，联合用药组和MGF24E组支架的细胞相容性优于rhBMP-2组和阴性对照支架。
6.建立了兔桡骨15mm节段性临界骨缺损模型，以PBS为阴性对照、80μgrhBMP-2为阳性对照，从影像学、组织学和生物力学三方面考察200μg MGF24E和200μg MGF24E+80μg rhBMP-2联合用药对临界骨缺损修复的作用；依据成骨相关蛋白OPN和新生血管标记蛋白CD31的表达规律分析MGF24E和MGF24E+rhBMP-2联合用药的临界骨缺损修复机制。
①影像学结果显示，联合用药修复临界骨缺损的骨皮质桥接愈合时间为12周，骨痂桥接的临床愈合时间为4周，早于rhBMP-2组；rhBMP-2组样本愈合经历了过度成骨，新生骨快速吸收（尺桡骨融合现象）的过程，联合用药完全逆转了rhBMP-2骨修复异常的趋势；术后12周MGF24E组虽只有50%样本达到骨皮质愈合标准，但远多于阴性对照组；各组4-12周骨改建时期影像学修复情况符合时间的线性函数关系；
②组织学结果显示，术后初期联合用药组和MGF24E组支架的组织相容性优于rhBMP-2组和阴性对照支架；组织学分析显示联合用药组愈合情况最好，优于rhBMP-2组和MGF24E组，表现出联合用药的协同增效作用；各组组织学愈合情况满足时间的对数函数关系；
③生物力学试验结果显示，虽然各用药组缺损区重建组织骨质连续，但其力学性能仍低于正常桡骨组织；各用药组样本刚度依次为：联合用药组> rhBMP-2组> MGF24E组，样本弯曲强度刚度依次为：联合用药组>MGF24E组> rhBMP-2，组联合用药组在力学性能上表现出协同增效作用；
④修复机制研究结果显示，用药组骨缺损愈合方式为软骨内成骨；MGF24E组能提早启动骨基质合成和血运恢复，因此含有MGF24E的联合用药组样本新骨形成量多于rhBMP-2组，但MGF24E的这种促进作用主要体现在术后初期，随着rhBMP-2组样本血运的逐渐恢复，联合用药组和rhBMP-2组间新骨形成差异逐渐减小。
综上所述，MGF24E既能显著地促血管新生，又能在一定程度上促进体内成骨，因此MGF24E+rhBMP-2联合用药能使临界骨缺损提早愈合，其修复质量也优于rhBMP-2或MGF24E单给药，表现出协同增效作用，并且联合用药能解决rhBMP-2成骨异常问题，为后续骨缺损修复的研究奠定了基础。
Large resections around bone tumors, complications after bone fracture, orinflammatory bone diseases often result in major bone loss or bone defect. Bone defecthealing is one of the challenges of orthopaedic surgery, for which bone grafts are widelyused. However, bone grafts have several drawbacks and are far from ideal, including thehigh donor-site morbidity and a high complication rate. So the new alternatives areneeded to satisfy clinical treatment. In search of alternative therapies, growth factorsgain a significant importance. At present, the growth factors approvalled by FDA for theclinical bone repair treatments are mainly BMPs family protein, in which rhBMP-2isthe mosr active one. But there are some drawbacks for rhBMP-2treatment: high cost,ectopic bone, excessive bone, and stimulate broken bone cell differentiation leading tofast absorption after the new bone formation. We aims at the problems of rhBMP-2treatment in the clinical application, and try to search an alternative factor for rhBMP-2,or one factor can have synergistic effect with rhBMP-2on bone repair. In recent year,C-terminal24-a.a. peptide of mechano growth factor, MGF24E gained an increasingattention because of its regenerative effects and the tissue-protective actions. MGF is theinitial splice-variant of IGF-1when tissue or cells suffer from damage.According to theprevious research results of our team on MGF24E, the studies are undertaken toinvestigate the roles and mechanism of mechano growth factor on bone injury repair, inorder to solve the problem of rhBMP-2on the clinical treatment. The main works andconclusions are included as follows:
1. The1mm or so bone fractures in radius of SD rats were prepared, and MGFprotein expression in jury was analised by Western blot with rabbit anti-MGF/MGF24Epolyclonal antibody. Results showed that MGF protein expression one daypostoperatively, rise to higher value, to peak3days postoperatively, and then slightlyfall down but still maintain a high expression in a relatively long period.
2.5mm segmental fracture in the radius of rabbit was treated by exogenousMGF24E injection, and the repair effects were assessed as radiographical andhistological observation and quantitative analysis.
①The clinical-healing time in high-dose treatment group was shorter2weeksthan that in the control group and low-dose treatment group. The regenerating bones inhigh-dose treatment group are tube-shape bone with reopening of the medullary cavity and the repairment was significantly better than the control group and low-dosetreatment group.
②The high-dose treatment significantly improves angiogenesis of regeneratingbone around defective areas, new blood vessels of high-dose group are more2foldsthan that of the control group.
3. The roles of MGF24E on angiogenesis and the underlying mechanisms wereinvestigated. The cell proliferation, migration, and tubulogenesis of the human vascularendothelial EA.hy926cells co-treated with2%serum and MGF24E were determined toassess angiogenesis in comparison with100ng/ml of VEGF165-positive control orvehicle control (phosphate-buffered saline).
①The pro-proliferation capacity of10ng/mL MGF24E was same to that of100ng/mL VEGF165. MGF24E promoted cell proliferation by inducing the cell cycleS-phase entry and transition to DNA replication and via an MAPK/ERK-dependentsignaling pathway.
②The capacities of MGF24E on promoting migration and tubulogenesis wasstronger than those of the control, but weaker than VEGF165.MGF24E-induced highermigratory activity and tubulogenesis partially involved ERK-signaling pathway.
③The suppression of vascular endothelial growth factor and angiopoietin-Iexpressions by2%serum starvation was reversed by the addition of10ng/ml ofMGF24E in2%serum medium, and the MGF24E-upregulated VEGF and Ang-I werehigher than those of the cells without serum starvation, which partially involvedERK-signaling pathway.
4. The effects of MGF24E on the osteogentic behaviors of primary osteoblastsand the underlying mechanisms were investigated. The cell proliferation, migration,ALP activity, mineralization and calcium secretion expression of primary osteoblastsco-treated with7%serum and dual growth factors (containing25ng/mL MGF24E+50ng/mL rhBMP-2) were determined to assess physiological behaviors of primaryosteoblasts in comparison with100ng/mL VEGF165+50ng/mL rhBMP-2-positive controlor vehicle control (phosphate-buffered saline) or25ng/mL MGF24E or50ng/mLrhBMP-2, and meanwhile the molecular mechanism of osteogenesis in vitro wereinvestigated.
①The pro-proliferation capacity of MGF24E was stronger than that of rhBMP-2or vehicle control. The activities of dual treatment (MGF24E+rhBMP-2) on osteoblastsproliferation, migration, osteodifferentiation and mineralization were higher than those of MGF24E treatment or rhBMP-2treatment. Although pro-migration activity ofpositive control (VEGF165+rhBMP-2) was higher than that of rhBMP-2treatment, theactivities of positive control on proliferation, osteodifferentiation and mineralizationwere lower than those of rhBMP-2treatment.
②The synergistic effect of dual treatment mainly involved the upregulations ofRunx2and OPN mRNA expressions
5. The composite scaffold containing different growth factors was constructedwith the carrier of absorbable gelatin sponge by the manner of negative pressurephysical adsorption at4℃,and the morphology structure, porosity of scaffold, releasecharacteristics of growth factors in vitro and cells compatibility of scaffold wereevaluated.
①The results of scanning electron microscopy (SEM) showed that growth factordistributed in scaffold appearing as a ball-shape, pore size and porosity can satisfy thegrowth of osteoblast and new capillary;
②The results of release characteristics in vitro showes that sudden release ofdifferent growth factores was observed in the initial period; MGF24E release was fastestand rhBMP-2release was the slowest. the release characteristics of MGF24E can meetthe demand of injury repair, and the release characteristics of rhBMP-2can meet theway of high concentration at one-time in clinical treatment;
③The results of cell activity of different scaffolds showed, different growthfactors led to different cytocompatibilities, and cytocompatibilities of dual treatmentgroup and MGF24E treatment group were better than those of rhBMP-2group andvehicle control.
6. The15mm segmental critical bone defect models in radius of rabbits wereestablished. The radiographical investigation, histological investigation andbiomechanics measurement were taken to evaluate the bone-defect repair capability ofdual treatment (containing200μg/mL MGF24E+80μg/mL rhBMP-2), in comparisonwith80μg/mL rhBMP-2-positive control or vehicle control (phosphate-buffered saline)or200μg/mL MGF24E, and meanwhile the repair mechanism of bone-defect in vitrowere investigated according to the expressions of OPN and CD31.
①The radiographical results showed, that healing time of cortex-bridging in dualtreatment groups was12weeks, and clinical-healing time of callus-bridging in dualtreatment groups was4weeks,which was earlier than that of rhBMP-2treatment groups;after samples of rhBMP-2treatment group appeared excessive bone, regenerating bone appeared fast absorption (radius-ulna fusion phenomenon), but dual treatmentcompletely reversed the abnormal bone-healing phenomena induced by rhBMP-2,suggesting a synergistic effect; Although during12weeks of repair period, only halfsamples of MGF24E treatment group appeared cortex-bridging, but were still far morethan that of the negative control group; the radiographical scores of different treatmentsmeted the linear function;
②The histological results showed, the differences of histocompatibilities ofdifferent growth factors treatment scaffolds were observed in the initial periodpostoperatively, and histocompatibilities of dual treatment scaffold and MGF24Etreatment scaffold were better than that of rhBMP-2treatment scaffold and the vehiclescaffold; regenerating bone mass and histological scores showed that bone-healingcapability of dual treatment group was the best, better than rhBMP-2treatment groupand MGF24E treatment group, suggesting the synergistic effect; The histological scoresmeted the logarithm function, according with the S curve law of biological communitiesgrowth.
③The biomechanical test results showed that, even if the regenerating bone ofdifferent treatment groups appeared bone mass bridging, mechanical properties ofregenerating bone were still lower than native radius; the stiffness and bending intensityof regenerating bone samples in MGF24E treatment group were about a third of thoseof native radius; the stiffness of rhBMP-2treatment group was nearly the half of that ofnative radius, and the bending intensity of rhBMP-2treatment group is about1/5of thatof native radius, the stiffness and bending intensity of dual treatment group were64%and54%of those of native radius respectively, suggesting the synergistic effect;
④The results of osteogenesis mechanism showed that, bone-defect repair modeof different growth treatments was endochondral ossification; the treatments containingMGF24E can induce early bone matrix synthesis and blood supply recovery, thus thenew bone formation of samples in dual treatment group was better than that of samplesin rhBMP-2treatment group, but the capability of MGF24E on promoting bone matrixsynthesis and blood supply recovery displayed in the initial period postoperatively, asthe gradual blood supply recovery of samples in rhBMP-2treatment group,thedifferences of dual treatment group and rhBMP-2treatment group in new boneformation was smaller.
To sum up, MGF24E not only can significantly promote angiogenesis, but also canpromote bone formation in vivo, so dual treatment of MGF24E and rhBMP-2can promote critical bone-defect healing in advance, and its repair quality was better thanregenerating bone in rhBMP-2or MGF24E treatment groups, suggesting the synergisticeffect; The key was that but dual treatment completely reversed the abnormalbone-healing phenomena induced by rhBMP-2, contributing to some further studies asan therapy of critical bone defect healing and showing an significance for clinicaltreatment.
1.在制备了1mm左右间隙的SD大鼠桡骨开放性骨折模型的基础上，利用前期制备的MGF/MGF24E兔抗人多抗，采用Western blot技术检测和分析大鼠骨折修复组织中MGF蛋白的表达情况。结果显示截骨损伤组织中MGF蛋白表达在术后1天即上升到较高值，术后第3天达到最高值，随后表达量虽有下降但仍维持较高表达。
2.从影像学和组织学的观察及分析角度，综合评价外源MGF24E注射修复兔桡骨节段性骨折的效果。
①高剂量MGF24E修复兔桡骨5mm节段性骨折的临床愈合时间，较MGF24E低剂量组和阴性对照组提早2周；MGF24E高剂量组重建骨组织为髓腔贯通的管状骨，其修复情况明显好于MGF24E低剂量组和阴性对照组；
②高剂量MGF24E能显著促进骨折重建组织的血管新生，组织学评分结果显示高剂量组样本新生血管数为阴性对照组的2倍以上。
3.采用2%FBS血清饥饿细胞模型，以PBS为阴性对照、100ng/mL VEGF165为阳性对照，考察MGF24E对人静脉内皮EA.hy926细胞增殖、迁移和体外管腔形成的影响；以PBS为阴性对照，10%FBS正常培养条件为阳性对照，考察10ng/mLMGF24E对人静脉内皮EA.hy926细胞VEGF和Ang-I的mRNA及蛋白表达的影响；同时还研究了MAPK/ERK通路在MGF24E影响血管内皮细胞体外血管化细胞行为及其分子机制中的作用。
①10ng/mL MGF24E促细胞增殖能力等效于100ng/mLVEGF165，MGF24E促细胞增殖是通过增加S期细胞数目和促进细胞DNA合成的方式完成，并完全依赖MAPK/ERK信号通路；
②MGF24E促细胞迁移、管腔形成的能力明显强于PBS阴性对照，但弱于VEGF165，并发现MAPK/ERK通路部分参与了MGF24E促细胞迁移、管腔形成等事件；
③MGF24E能逆转血清饥饿引起的正常生长细胞VEGF和Ang-I表达下调的趋势，其上调量高于正常培养组的表达量，并发现MAPK/ERK通路部分参与了MGF24E上调VEGF和Ang-I表达的整个过程。
4.采用7%FBS血清饥饿细胞模型，以PBS为阴性对照、100ng/mLVEGF165+50ng/mL rhBMP-2的联合用药为阳性对照，25ng/mL MGF24E、50ng/mLrhBMP-2单给药为联合用药组的增效参照，考察25ng/mL MGF24E+50ng/mLrhBMP-2联合用药对原代成骨细胞增殖、迁移、分化早期指标ALP活性以及矿化钙分泌量表达的影响，并研究其分子机制。
①MGF24E促细胞增殖能力强于rhBMP-2和PBS，相对于MGF24E或rhBMP-2单独给药，MGF24E+rhBMP-2联合用药对促进细胞DNA合成、迁移和分化矿化有协同增效作用；阳性对照除对促进细胞迁移有协同增效作用外，在其他成骨相关细胞生理行为上均表现为拮抗抑制作用；
②MGF24E+rhBMP-2联合用药在细胞分化矿化事件上的协同增效作用主要涉及调控成骨信号通路的转录因子Runx2基因和骨基质形成的OPN基因表达上调。
5.以可吸收明胶海绵(Absorbable Gelatin Sponge, AGS)作为载体，采用低温负压物理吸附法制备4种不同给药方案的复合生长因子支架，并对复合支架的形貌结构、孔隙率、生长因子的体外释放以及支架细胞相容性等性能进行评价。
①扫描电镜结果显示，生长因子成球状分布于复合AGS支架内部，支架孔径和孔隙率能满足成骨细胞长入；
②体外释放结果显示，不同复合支架中生长因子的体外释放均发生突释现象，其中MGF24E释放最快，rhBMP-2释放最慢，生长因子的释放规律可满足机体对MGF24E需求以及临床上rhBMP-2的一次性高浓度给药方式；
③复合支架中成骨细胞活性实验结果显示，各组复合支架表现出细胞相容性差异，联合用药组和MGF24E组支架的细胞相容性优于rhBMP-2组和阴性对照支架。
6.建立了兔桡骨15mm节段性临界骨缺损模型，以PBS为阴性对照、80μgrhBMP-2为阳性对照，从影像学、组织学和生物力学三方面考察200μg MGF24E和200μg MGF24E+80μg rhBMP-2联合用药对临界骨缺损修复的作用；依据成骨相关蛋白OPN和新生血管标记蛋白CD31的表达规律分析MGF24E和MGF24E+rhBMP-2联合用药的临界骨缺损修复机制。
①影像学结果显示，联合用药修复临界骨缺损的骨皮质桥接愈合时间为12周，骨痂桥接的临床愈合时间为4周，早于rhBMP-2组；rhBMP-2组样本愈合经历了过度成骨，新生骨快速吸收（尺桡骨融合现象）的过程，联合用药完全逆转了rhBMP-2骨修复异常的趋势；术后12周MGF24E组虽只有50%样本达到骨皮质愈合标准，但远多于阴性对照组；各组4-12周骨改建时期影像学修复情况符合时间的线性函数关系；
②组织学结果显示，术后初期联合用药组和MGF24E组支架的组织相容性优于rhBMP-2组和阴性对照支架；组织学分析显示联合用药组愈合情况最好，优于rhBMP-2组和MGF24E组，表现出联合用药的协同增效作用；各组组织学愈合情况满足时间的对数函数关系；
③生物力学试验结果显示，虽然各用药组缺损区重建组织骨质连续，但其力学性能仍低于正常桡骨组织；各用药组样本刚度依次为：联合用药组> rhBMP-2组> MGF24E组，样本弯曲强度刚度依次为：联合用药组>MGF24E组> rhBMP-2，组联合用药组在力学性能上表现出协同增效作用；
④修复机制研究结果显示，用药组骨缺损愈合方式为软骨内成骨；MGF24E组能提早启动骨基质合成和血运恢复，因此含有MGF24E的联合用药组样本新骨形成量多于rhBMP-2组，但MGF24E的这种促进作用主要体现在术后初期，随着rhBMP-2组样本血运的逐渐恢复，联合用药组和rhBMP-2组间新骨形成差异逐渐减小。
综上所述，MGF24E既能显著地促血管新生，又能在一定程度上促进体内成骨，因此MGF24E+rhBMP-2联合用药能使临界骨缺损提早愈合，其修复质量也优于rhBMP-2或MGF24E单给药，表现出协同增效作用，并且联合用药能解决rhBMP-2成骨异常问题，为后续骨缺损修复的研究奠定了基础。
Large resections around bone tumors, complications after bone fracture, orinflammatory bone diseases often result in major bone loss or bone defect. Bone defecthealing is one of the challenges of orthopaedic surgery, for which bone grafts are widelyused. However, bone grafts have several drawbacks and are far from ideal, including thehigh donor-site morbidity and a high complication rate. So the new alternatives areneeded to satisfy clinical treatment. In search of alternative therapies, growth factorsgain a significant importance. At present, the growth factors approvalled by FDA for theclinical bone repair treatments are mainly BMPs family protein, in which rhBMP-2isthe mosr active one. But there are some drawbacks for rhBMP-2treatment: high cost,ectopic bone, excessive bone, and stimulate broken bone cell differentiation leading tofast absorption after the new bone formation. We aims at the problems of rhBMP-2treatment in the clinical application, and try to search an alternative factor for rhBMP-2,or one factor can have synergistic effect with rhBMP-2on bone repair. In recent year,C-terminal24-a.a. peptide of mechano growth factor, MGF24E gained an increasingattention because of its regenerative effects and the tissue-protective actions. MGF is theinitial splice-variant of IGF-1when tissue or cells suffer from damage.According to theprevious research results of our team on MGF24E, the studies are undertaken toinvestigate the roles and mechanism of mechano growth factor on bone injury repair, inorder to solve the problem of rhBMP-2on the clinical treatment. The main works andconclusions are included as follows:
1. The1mm or so bone fractures in radius of SD rats were prepared, and MGFprotein expression in jury was analised by Western blot with rabbit anti-MGF/MGF24Epolyclonal antibody. Results showed that MGF protein expression one daypostoperatively, rise to higher value, to peak3days postoperatively, and then slightlyfall down but still maintain a high expression in a relatively long period.
2.5mm segmental fracture in the radius of rabbit was treated by exogenousMGF24E injection, and the repair effects were assessed as radiographical andhistological observation and quantitative analysis.
①The clinical-healing time in high-dose treatment group was shorter2weeksthan that in the control group and low-dose treatment group. The regenerating bones inhigh-dose treatment group are tube-shape bone with reopening of the medullary cavity and the repairment was significantly better than the control group and low-dosetreatment group.
②The high-dose treatment significantly improves angiogenesis of regeneratingbone around defective areas, new blood vessels of high-dose group are more2foldsthan that of the control group.
3. The roles of MGF24E on angiogenesis and the underlying mechanisms wereinvestigated. The cell proliferation, migration, and tubulogenesis of the human vascularendothelial EA.hy926cells co-treated with2%serum and MGF24E were determined toassess angiogenesis in comparison with100ng/ml of VEGF165-positive control orvehicle control (phosphate-buffered saline).
①The pro-proliferation capacity of10ng/mL MGF24E was same to that of100ng/mL VEGF165. MGF24E promoted cell proliferation by inducing the cell cycleS-phase entry and transition to DNA replication and via an MAPK/ERK-dependentsignaling pathway.
②The capacities of MGF24E on promoting migration and tubulogenesis wasstronger than those of the control, but weaker than VEGF165.MGF24E-induced highermigratory activity and tubulogenesis partially involved ERK-signaling pathway.
③The suppression of vascular endothelial growth factor and angiopoietin-Iexpressions by2%serum starvation was reversed by the addition of10ng/ml ofMGF24E in2%serum medium, and the MGF24E-upregulated VEGF and Ang-I werehigher than those of the cells without serum starvation, which partially involvedERK-signaling pathway.
4. The effects of MGF24E on the osteogentic behaviors of primary osteoblastsand the underlying mechanisms were investigated. The cell proliferation, migration,ALP activity, mineralization and calcium secretion expression of primary osteoblastsco-treated with7%serum and dual growth factors (containing25ng/mL MGF24E+50ng/mL rhBMP-2) were determined to assess physiological behaviors of primaryosteoblasts in comparison with100ng/mL VEGF165+50ng/mL rhBMP-2-positive controlor vehicle control (phosphate-buffered saline) or25ng/mL MGF24E or50ng/mLrhBMP-2, and meanwhile the molecular mechanism of osteogenesis in vitro wereinvestigated.
①The pro-proliferation capacity of MGF24E was stronger than that of rhBMP-2or vehicle control. The activities of dual treatment (MGF24E+rhBMP-2) on osteoblastsproliferation, migration, osteodifferentiation and mineralization were higher than those of MGF24E treatment or rhBMP-2treatment. Although pro-migration activity ofpositive control (VEGF165+rhBMP-2) was higher than that of rhBMP-2treatment, theactivities of positive control on proliferation, osteodifferentiation and mineralizationwere lower than those of rhBMP-2treatment.
②The synergistic effect of dual treatment mainly involved the upregulations ofRunx2and OPN mRNA expressions
5. The composite scaffold containing different growth factors was constructedwith the carrier of absorbable gelatin sponge by the manner of negative pressurephysical adsorption at4℃,and the morphology structure, porosity of scaffold, releasecharacteristics of growth factors in vitro and cells compatibility of scaffold wereevaluated.
①The results of scanning electron microscopy (SEM) showed that growth factordistributed in scaffold appearing as a ball-shape, pore size and porosity can satisfy thegrowth of osteoblast and new capillary;
②The results of release characteristics in vitro showes that sudden release ofdifferent growth factores was observed in the initial period; MGF24E release was fastestand rhBMP-2release was the slowest. the release characteristics of MGF24E can meetthe demand of injury repair, and the release characteristics of rhBMP-2can meet theway of high concentration at one-time in clinical treatment;
③The results of cell activity of different scaffolds showed, different growthfactors led to different cytocompatibilities, and cytocompatibilities of dual treatmentgroup and MGF24E treatment group were better than those of rhBMP-2group andvehicle control.
6. The15mm segmental critical bone defect models in radius of rabbits wereestablished. The radiographical investigation, histological investigation andbiomechanics measurement were taken to evaluate the bone-defect repair capability ofdual treatment (containing200μg/mL MGF24E+80μg/mL rhBMP-2), in comparisonwith80μg/mL rhBMP-2-positive control or vehicle control (phosphate-buffered saline)or200μg/mL MGF24E, and meanwhile the repair mechanism of bone-defect in vitrowere investigated according to the expressions of OPN and CD31.
①The radiographical results showed, that healing time of cortex-bridging in dualtreatment groups was12weeks, and clinical-healing time of callus-bridging in dualtreatment groups was4weeks,which was earlier than that of rhBMP-2treatment groups;after samples of rhBMP-2treatment group appeared excessive bone, regenerating bone appeared fast absorption (radius-ulna fusion phenomenon), but dual treatmentcompletely reversed the abnormal bone-healing phenomena induced by rhBMP-2,suggesting a synergistic effect; Although during12weeks of repair period, only halfsamples of MGF24E treatment group appeared cortex-bridging, but were still far morethan that of the negative control group; the radiographical scores of different treatmentsmeted the linear function;
②The histological results showed, the differences of histocompatibilities ofdifferent growth factors treatment scaffolds were observed in the initial periodpostoperatively, and histocompatibilities of dual treatment scaffold and MGF24Etreatment scaffold were better than that of rhBMP-2treatment scaffold and the vehiclescaffold; regenerating bone mass and histological scores showed that bone-healingcapability of dual treatment group was the best, better than rhBMP-2treatment groupand MGF24E treatment group, suggesting the synergistic effect; The histological scoresmeted the logarithm function, according with the S curve law of biological communitiesgrowth.
③The biomechanical test results showed that, even if the regenerating bone ofdifferent treatment groups appeared bone mass bridging, mechanical properties ofregenerating bone were still lower than native radius; the stiffness and bending intensityof regenerating bone samples in MGF24E treatment group were about a third of thoseof native radius; the stiffness of rhBMP-2treatment group was nearly the half of that ofnative radius, and the bending intensity of rhBMP-2treatment group is about1/5of thatof native radius, the stiffness and bending intensity of dual treatment group were64%and54%of those of native radius respectively, suggesting the synergistic effect;
④The results of osteogenesis mechanism showed that, bone-defect repair modeof different growth treatments was endochondral ossification; the treatments containingMGF24E can induce early bone matrix synthesis and blood supply recovery, thus thenew bone formation of samples in dual treatment group was better than that of samplesin rhBMP-2treatment group, but the capability of MGF24E on promoting bone matrixsynthesis and blood supply recovery displayed in the initial period postoperatively, asthe gradual blood supply recovery of samples in rhBMP-2treatment group,thedifferences of dual treatment group and rhBMP-2treatment group in new boneformation was smaller.
To sum up, MGF24E not only can significantly promote angiogenesis, but also canpromote bone formation in vivo, so dual treatment of MGF24E and rhBMP-2can promote critical bone-defect healing in advance, and its repair quality was better thanregenerating bone in rhBMP-2or MGF24E treatment groups, suggesting the synergisticeffect; The key was that but dual treatment completely reversed the abnormalbone-healing phenomena induced by rhBMP-2, contributing to some further studies asan therapy of critical bone defect healing and showing an significance for clinicaltreatment.
引文
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