Research On Mechanism Of Strontium-substituted Sub-micron Bioactive Glasses For Improved Bone Repair

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.