【作者】 张文；

【导师】 陈晓峰；

【作者基本信息】 华南理工大学 ， 生物医学工程， 2017， 博士

【摘要】 生物活性玻璃因具有良好的骨传导性和骨诱导性,在骨修复领域具有良好的应用前景。然而,目前临床上应用的生物活性玻璃多是由熔融法制备,存在易团聚、结构和尺寸不可控、形貌不规则等问题,这些问题影响了生物活性玻璃的离子释放,导致其在骨缺损修复中的应用受限。本课题组胡庆等通过以锶（Sr）替代组分中的部分钙（Ca）,制备出了形貌规则、尺寸可调、具有良好分散性的新型掺Sr微纳米生物活性玻璃,但掺Sr微纳米生物活性玻璃促进成骨的机制,以及其体内的骨修复能力尚且未知,因此,在这种掺Sr微纳米生物活性玻璃应用于临床骨修复前,需要对其促进骨修复的机制进行深入研究。目前评价骨生物材料体外骨修复效果的主要方法是研究材料是否能直接刺激具有成骨能力细胞的成骨分化。然而,许多体外评价时能成功诱导成骨的材料在用于体内研究时,其结果会出现与体外研究不一致的表现,这意味着我们对材料介导成骨的机制理解得并不透彻。近年来越来越多的研究证明免疫系统在骨修复中起着关键的作用。骨生物材料作为异物,一旦植入到宿主体内即可被免疫系统识别并触发显著的免疫反应,进而影响其骨修复效果。这提示我们在研发骨生物材料时,除了应用传统的方法来评估其骨修复能力外,还需要研究材料所诱导的免疫微环境对骨修复的影响。本文制备了具有亚微米结构的生物活性玻璃微球（SBG）和掺Sr的亚微米生物活性玻璃微球（Sr-SBG）,并系统地评估了它们的骨修复作用及其作用机制。首先,使用传统的骨生物材料成骨能力评价方法,研究了材料促进骨髓间充质干细胞向成骨分化的作用及其分子机制;其次,阐明了材料对破骨细胞分化的影响及其分子机制;再次,探讨了材料诱导的免疫微环境以及该免疫微环境对成骨分化和破骨分化的影响;最后,通过对比体内实验与体外实验的结果,揭示了Sr-SBG促进骨修复的作用机制。主要研究工作和结论如下:（1）通过溶胶凝胶技术与模板自组装技术相结合的方法,制备了具有良好单分散性的SBG,并通过以Sr替代部分Ca,制备了Sr-SBG。研究了Sr-SBG和SBG浸提液以及与Sr-SBG浸提液具有相同Sr离子浓度的SrCl₂对小鼠骨髓间充质干细胞（mMSCs）向成骨分化的作用,结果表明:Sr-SBG相较于SBG和SrCl₂具有更强的促成骨分化能力。进一步对Sr-SBG促进mMSCs成骨分化的分子机制进行研究,结果显示:SBG能够通过Wnt/β-catenin信号通路促进成骨分化,而Sr-SBG能够通过NFATc信号通路以及Wnt/β-catenin信号通路的协同作用,在Sr-SBG的促成骨分化中起作用。抑制NFATc信号通路能够完全抑制SrCl₂的促成骨分化作用,部分抑制Sr-SBG的促成骨分化效果,而对SBG的促成骨分化作用没有明显影响。（2）考察了SBG和Sr-SBG两种玻璃的浸提液以及与Sr-SBG浸提液具有相同Sr离子浓度的SrCl₂对核因子-κB受体活化因子配基（RANKL）诱导的RAW264.7细胞向破骨分化的作用,结果表明:Sr-SBG相较于SBG和SrCl₂具有更强的抑制破骨分化的能力。进一步对Sr-SBG抑制RANKL诱导的RAW264.7细胞破骨分化的分子机制进行研究,结果发现:Sr-SBG、SBG和SrCl₂均能抑制MAPK信号通路中的p38通路,其中Sr-SBG对p38通路的抑制作用最大,而三者对MAPK信号通路中的JNK和ERK1/2通路没有明显的抑制作用。另外,Sr-SBG和SrCl₂均能上调IκB-α的表达,从而通过抑制NF-κB信号通路来达到抑制破骨分化的作用。因此,Sr-SBG可能通过抑制MAPK下游的p38信号通路和抑制NF-κB信号通路来抑制破骨分化。（3）通过研究Sr-SBG对巨噬细胞极化的影响来明确Sr-SBG诱导的免疫微环境是否有利于成骨。结果发现:Sr-SBG能诱导巨噬细胞向M2极化,并且能抑制促炎症因子IL6的分泌,抑制促炎症反应基因IL1β和iNOS的表达;促进抑炎症反应基因IL1ra,ArginaseⅠ的表达,促进促成骨分化基因BMP2的表达。用Sr-SBG诱导的巨噬细胞分泌产物对mMSCs的成骨分化和RAW264.7的破骨分化作用进行研究。结果发现:Sr-SBG诱导的巨噬细胞分泌产物能够促进成骨分化和抑制破骨分化。同时,进一步研究了Sr-SBG诱导的巨噬细胞条件培养液对mMSCs反向调节巨噬细胞破骨分化的能力,结果显示:在Sr-SBG诱导的巨噬细胞分泌产物作用下,mMSCs的OPG基因表达显著上调,而RANKL基因表达有所下调。且OPG/RANKL的比值相较于SBG组和Control组明显上升,表现出抑制破骨分化的趋势。（4）为了验证体外细胞实验的结果是否与体内实验结果一致,进行了体内皮下埋植和原位骨缺损修复实验。ELISA结果表明:Sr-SBG植入皮下后其周围组织的促炎症因子IL6的分泌较SBG明显下降。免疫组化染色结果显示:Sr-SBG周围M2型巨噬细胞标记分子CD206和Arginase I较SBG组多,说明Sr-SBG周围M2型巨噬细胞浸润较SBG明显增多;Sr-SBG周围M1型巨噬细胞标记分子NOS2浸润较SBG少,说明Sr-SBG周围M1型巨噬细胞浸润较SBG明显减少。Masson染色结果发现:Sr-SBG的体内成骨能力优于SBG,且从HE染色结果可知:Sr-SBG组相较于SBG组具有更少的纤维组织形成。对破骨细胞的TRAP染色结果表明:在材料植入初期,Sr-SBG和SBG材料周围均有大量的破骨细胞存在,而在骨修复后期,Sr-SBG组比SBG组的破骨细胞数量明显减少。对材料促进成骨的体内分子机制进行免疫组化研究,结果发现:Sr-SBG和SBG植入后均能促进细胞表达β-catenin,且植入Sr-SBG的骨缺损处细胞表达出更大量的β-catenin。同样地,Sr-SBG的植入也能促进细胞大量表达NFATc1,该结果与体外研究的结果具有一致性。综上所述,Sr-SBG一方面能通过释放的Sr离子与Si离子协同作用,激活mMSCs中的Wnt/β-catenin信号通路和NFATc信号通路促进成骨分化,抑制RAW264.7细胞中的p38信号通路和NFκB信号通路来抑制破骨分化,另一方面还能通过促进巨噬细胞适度的M2极化,创造有利于成骨的免疫微环境,来促进成骨分化和抑制破骨分化,从而促进骨修复。本论文阐明了Sr-SBG促进骨修复的机制,解决了其应用于骨组织修复的关键科学问题,对进一步优化骨生物材料以及更好地预测材料的骨修复效果具有重要的指导意义。更多还原

【Abstract】 As bioactive glass（BG）has the properties of bone conductivity and inductivity,it has a bright future in the field of bone repair.However,the current clinical applied BG prepared by melting method has the problem of easy to agglomerate,uncontrolled structure and size and irregular shape,thus affecting the ion release of BG,leading to its limited application in bone repair.Previous research by Hu Qing in our group has developed strontium（Sr）substituted micro/nano bioactive glass with regular shape,controlled structure,composition and size by replacing partial calcium（Ca）with Sr.However,the mechanism of osteogenesis mediated by Sr-substituted bioactive glass and its bone repair ability in vivo are still unknown.Therefore,the mechanism of enhanced bone repair induced by Sr-substituted micro/nano bioactive glass should be researched into before the clinical application of Sr-substituted bioactive glass for bone repair.At present,the main method to evaluate the osteogenesis of bone biomaterials in vitro is to investigate whether the material could directly induce osteogenesis from osteoblastic cells.However,when used for in vivo study,the results of many materials that can successfully induce osteogenesis in vitro would be inconsistent with in vitro studies,which means that the mechanisms of material mediated bone formation is not well-understood.Recently,the immune system is found closely related to bone repair.As a foreign body,once implanted into the host body,bone biomaterials can be identified by the immune system and trigger a significant immune response,thus affecting the bone repair effect.Hence,in the development of bone biomaterials,in addition to using traditional method to evaluate bone repair capacity,the effect of local immune environment induced by materials on bone repair should also be studied.In this work,we have prepared sub-micron bioactive glass microspheres（SBG）and strontium substituted sub-micron bioactive glass microspheres（Sr-SBG）,and systematic evaluation has been performed to determine whether Sr-SBG could promote bone repair.Firstly,we investigated the osteogenesis effect and molecular mechanism of materials on bone marrow mesenchymal stem cells using the traditional osteogenic capacity evaluation method.Secondly,the osteocalstogenesis effect and molecular mechanism of materials on macraphage were illuminated.Third,the immune micro-environment induced by materials and the effect of materials induced micro-environment on osteogenesis and osteoclastogenesis were discussed.Finally,the mechanism of bone repair effect mediated by Sr-SBG was identified through comparing results from in vitro and in vivo experiment.Main research work and conclusions are stated as follows:（1）The sub-micron bioactive glasses with good mono-dispersion property were prepared via combined sol-gel method and template self-assembly technology.Furthermore,we prepared strontium substituted sub-micron bioactive glasses by replacing partial Ca with Sr.The effect of Sr-SBG extracts,SBG extracts,and SrCl₂ with identical Sr ion concentration on osteogenic differentiation of mouse mesenchymal stem cells（m MSCs）was assessed comparatively.The results indicated that Sr-SBG had superior osteogenic differentiation capability than SBG and SrCl₂.Moreover,the molecular mechanism of Sr-SBG on promoting osteogenic differentiation of mMSCs has been investigated.The results showed that SBG could promote osteogenesis via Wnt/β-catenin signaling pathway,and Sr-SBG could improve osteogenesis by the synergistic effect of NFATc signaling pathway and Wnt/β-catenin signaling pathway.When NFATc signaling pathway was blocked,the enhanced osteogenesis induced by SrCl₂ was totally abrogated,and the improved osteogenesis mediated by Sr-SBG was partially inhibited.However,the promoted osteogenic differentiation capacity stimulated by SBG was unaffected.（2）The effect of Sr-SBG extracts,SBG extracts,and SrCl₂ with identical Sr ion concentration on Receptor Activator of Nuclear Factor-Kappa B Ligand（RANKL）induced osteoclastogenesis of RAW264.7 cells was evaluated comparatively.The results indicated that Sr-SBG had more profound effect in inhibiting osteocalstogenesis than SBG and SrCl₂.And the molecular mechanism of the inhibited effect mediated by Sr-SBG was also investigated.The results showed that the downstream p38 signaling pathway of MAPK was downregulated in SBG,Sr-SBG and SrCl₂ group,with Sr-SBG being the most significantly decreased.There were no obvious change in JNK and ERK1/2 signaling pathway among experimental groups.In addition,IκB-αwas upregulated in Sr-SBG and SrCl₂ group,suggesting that Sr-SBG might suppress osteocalstogenesis via blocking p38 signaling pathway and NFκB signaling pathway.（3）The problem of whether the immune micro-environment induced by Sr-SBG is benifical for osteogenesis was studied by the assessment of Sr-SBG on macrophage polarization.The results demonstrated that Sr-SBG could mediate M2 polarization of macrophage.The secretion of inflammatory cytokine IL6 was downregulated.The gene expressionsof inflammatoryIL1βandiNOSweredownregulated,whilethe anti-inflammatory gene expressions IL1ra and Arginase were upregulated.Meanwhile,the m RNA expression of BMP2,which can promote osteogenic differentiation,was also upregulated.Furthermore,the effect of Sr-SBG induced macrophage-conditioned medium on osteogenesis of mMSCs and osteoclastogenesis of RAW264.7 cells was also evaluated.The results showed that the secretion of macrophage induced by Sr-SBG could further promote osteogenesis and inhibit osteoclastogenesis.Moreover,examination of osteoclastogenesis induced by mMSCs production reacte to Sr-SBG stimulated macrophage-conditioned was studied.The results indicated that the secretion component of Sr-SBG stimulated macrophage-conditioned medium could reacte to macrophage through enhance the osteoclastogenesis-related gene expressions OPG,and decrease the RANKL expression.The OPG/RANKL ratio of mMSCs was greatly increased compared to SBG and Control group,which showed a tendency to inhibit osteoclastogensis.（4）In order to verify the in vitro results are consistent with the results of in vivo experiments,in vivo study was performed to through subcutaneous implantation,in situ bone defect repair experiments.The ELISA results showed that Sr-SBG implanted sample exhibited decrease secretion of inflammatory IL6 than SBG implanted sample.The immunohistochemistry results revealed that the M2 macrophage marker CD206 and Arginase I around Sr-SBG was more obvious than that around SBG,on the contrast,M1 macrophage marker NOS2 around Sr-SBG was much less that that around SBG.It demonstrated that M1macrophage around Sr-SBG was decreased,while M2 macrophage around Sr-SBG was increased.Masson staining results showed that bone formation around Sr-SBG was enhanced than SBG.H&E staining indicated that there were more inflammation infiltration around Sr-SBG implant than SBG implant.TRAP staining results revealed that the number of osteoclast was similar between SBG and Sr-SBG group on the early stage of implantation,while less osteoclast was observed in Sr-SBG group than that in SBG group on the late stage of bone repair.In vivo molecular mechanism of improved osteogenesis induced by Sr-SBG were examined by immunohistochemistry staining of NFATc1 andβ-catenin.The results demonstrated tissues around Sr-SBG group expressed more NFATc1 andβ-catenin than SBG,which was consistent with in vitro results.In conclusion,on one hand,Sr-SBG could not only improve osteogenesis of mMSCs through activation of Wnt/β-catenin signaling pathway and NFATc signaling pathway by the synergistic effect of released Si and Sr ions,but also inhibit RANKL-mediated osteoclastogenesis of RAW264.7 cells through suppressing p38 signaling pathway and NFκB signaling pathway.On the other hand,Sr-SBG could promote osteogenesis and inhibit osteoclastogenesis via stimulating macrophage toward moderate M2 polarization to create a favorable immune micro-environment.The obtained results clarified the mechanism of Sr-SBG on bone repair,and illuminated key scientific issues needed to be solved for the application of Sr-SBG in bone repair,which has guiding significance for the optimization of bone biomaterials and the prediction of bone repair effects of materials.更多还原