Nano-calcium-calibrator Rescues Inflammation-induced Mitochondria Dysfunction In Mesenchymal Stem Cells To Alleviate Inflammatory Associated Bone Diseases

BackgroundMesenchymal stem cells（MSCs）are widely used in disease treatment and tissue regeneration because of their multi-directional differentiation potential,wide source,and easy availability.Studies have shown that there are abnormal MSCs in the tissues of inflammatory bone diseases such as periodontitis and osteoarthritis,which is an important reason for the incurability of tissue damage.Mitochondria are the energy factories in cells with their own genetic material mt DNA and are also the main source of intracellular reactive oxygen species.As a result,mitochondria are of great significance in maintaining the differentiation potential of stem cells.Among the reasons to cause MSCs damage under chronic inflammation,it has been confirmed that the mitochondrial dysfunction in MSCs plays a critical role.However,there is still no effective method to cure the chronic inflammation associated bone diseases via physiologically rehabilitating the function of mitochondria and MSCs,due to the unclear mechanism.It is worth noting that some mitochondria can form highly associated structural regions with the endoplasmic reticulum,and their structural and functional coupling plays an important role in various cellular pathways.Recently,several studies demonstrate that the mitochondrial dysfunction comes from excess Ca²⁺transfer from endoplasm reticulum（ER）in obesity and Parkinson.Ca²⁺overload in mitochondria is considered to enhance intracellular production of reactive oxygen species（ROS）,which may damage relevant cells.However,it has not been reported whether mitochondrial structural and functional changes are involved in the manipulation of MSCs function in inflammatory microenvironment,and whether this process is mediated by endoplasmic reticulum mitochondrial coupling structure.Besides,Wnt/β-catenin signaling pathway is a classic pathway regulating the proliferation and differentiation of stem cells.In the inflammatory environment,Wnt/β-catenin pathway may be involved in the regulation of mitochondrial dynamics,thus affecting the differentiation ability of MSCs.In addition,the dysfunctional mitochondria continuously harm the function of cells,which makes the clearance of dysfunctional mitochondria even more important for the therapy of chronic inflammation associated bone disease.However,the current treatment strategies targeting mitochondrial related proteins,such as RNAi or peptides,can only stop the cell damage to a certain extent by blocking related pathways,which contribute few to sweep the destroyed mitochondria and to restore the function of damaged mitochondria.In this study,we used two kinds of tissue-derived MSCs,periodontal ligament mesenchymal stem cells（PDLSCs）and bone marrow mesenchymal stem cells（BMMSCs）,to explore the effect of inflammatory microenvironment on the mitochondrial function of MSCs,and designed nanoparticles to restore the mitochondrial homeostasis in MSCs,aiming at treating inflammatory bone diseases and promoting tissue regeneration.AimTo investigate the changes of mitochondrial structure and function in MSCs under inflammatory environment,clarify the mechanism of abnormal mitochondrial dynamics in MSCs caused by inflammation,and design functionalized nanoparticles targeting the corresponding mechanism to restore mitochondrial homeostasis in MSCs and alleviate inflammatory associated bone diseases.Methods1.Alterations of mitochondrial structure and function in MSCs under inflammatory environment:All Changes in the length,area and number of mitochondria and the degree of endoplasmic reticulum-mitochondrial structural coupling in MSCs under inflammatory microenvironment were observed and compared by transmission electron microscope and organelle fluorescence staining.In addition,mitochondrial calcium-regulated gene expression and mitofusins were detected by q RT-PCR.Mitochondrial Ca²⁺concentration in mitochondria was examined by mitochondrial Ca²⁺probe Rhod-2.Mitochondrial membrane potential was evaluated by JC-1 staining.Intracellular ROS concentration in MSCs under normal and inflammatory microenvironment were detected by the mitochondrial calcium ion probe,JC-1 staining and DFCA-H probe respectively.2.The regulation of mitophagy by Wnt/β-catenin pathway in MSCs in inflammatory environment.The expression of mitophagy-related proteins in MSCs under normal and inflammatory conditions was detected by Western Blot.Mitophagy activity and autophagosome formation in MSCs were observed by organelle fluorescence probe staining and confocal microscopy.After the expression ofβ-catenin was down-regulated or up-regulated by si RNA or overexpressed plasmid,the alterations of mitophagy were detected by Western Blot and confocal microscopy.3.Design and synthesis of nanoparticles METP/siβ-catenin.Functionalize mesoporous silica nanoparticles（MSN）with TPP,EGTA and siβ-catenin to target mitochondria,adsorb Ca²⁺around mitochondria and release si RNA to inhibit Wnt/β-catenin pathway.After the nanoparticles were synthesized,the co-localization of METP/siβ-catenin and mitochondria was detected by confocal microscopy,and the toxicity of the nanoparticles was measured by MTT assay.4.Regulation of mitochondria and MSCs functions by METP/siβ-catenin.After adding nanoparticles to the medium,mitochondria Ca²⁺concentration was detected by Rhod-2,andβ-catenin expression was analyzed by Western Blot.Transmission electron microscopy and confocal microscopy were applied to observe the effect of METP/siβ-catenin on mitochondrial dynamics.JC-1 staining and DCFA probe were used to detect the changes of mitochondrial membrane potential and intracellular ROS after the addition of nanoparticles.5.The therapeutic effect of METP/siβ-catenin on inflammatory bone diseases.Periodontitis models of SD rats were established by LPS application.Periodontal locally injection of METP/siβ-catenin was performed for treatment.The osteoarthritis models of C57 mice were established and treated by subchondral bone injection with METP/siβ-catenin.The joint structures were analyzed by micro-CT and saffron solid green staining.The expression of IP3R and MFN2 in MSCs in periodontal ligaments and subchondral bone was investigated by immunofluorescence staining of tissue sections.Results1.Inflammation causes excess Ca²⁺transfer from ER to mitochondria in MSCs derived from periodontitis and osteoarthritis patients.Compared with MSCs isolated from healthy individuals,the number,length,area and MAM coupling of mitochondria were increased in MSCs isolated from periodontitis and osteoarthritis patients or stimulated by TNF-α.Meanwhile,the calcium concentration in mitochondria was increased in the inflammatory microenviroment mediated by the increased expression of MAM coupling and calcium channel related molecules.The mitochondrial membrane potential was decreased accompanied with more ROS production in MSCs under inflammatory microenvironment.2.Activation of Wnt/β-catenin pathway inhibits mitophagy leading to the accumulation of damaged mitochondria in MSCs.Mitochondrial and lysosomal co-localization decreased in MSCs under inflammatory conditions,indicating impaired mitophagy activity.β-catenin blocks mitophagy by inhibiting the transformation from LC3-I to LC3-II in PDLSCs whileβ-catenin inhibits the expression of pink1 and parkin in BMMSCs.3.METP/siβ-catenin can restore mitochondria function in MSCs.METP/siβ-catenin can target the mitochondria of MSCs and adsorb excess calcium around mitochondria effectively,in order to prevent the formation of dysfuncitonal mitochondria.At the same time,siβ-catenin can be released to promote the elimination and degradation of the impaired mitochondria through mitophagy.Therefore,METP/siβ-catenin can restore mitochondrial structure,mitochondrial membrane potential and ROS production,and recover the osteogenic differentiation ability of MSCs.4.METP/siβ-catenin can alleviate inflammatory bone diseases.Nanoparticles injection can restore the height of alveolar bone in the periodontitis models,and reconstruct the articular surface cartilage structure in OA mice.The expression of MFN2 and IP3R2 in MSCs were decreased in tissue sections after treatment with functionalized nanoparticles.ConclusionsIn this study,we first showed that chronic inflammation leads to Ca²⁺transfer from the endoplasmic reticulum to mitochondria,leading to mitochondrial calcium overload and further mitochondrial damage.In addition,activation of the Wnt/β-catenin pathway inhibits mitochondrial autophagy under chronic inflammatory conditions,leading to the accumulation of damaged mitochondria in MSCs,thereby disrupting the differentiation of MSCs.Based on the mechanism of the new findings,we prepared cell microenvironment（esterase and low p H）responsive nanoparticles to capture the Ca²⁺around mitochondria in MSCs and and deliver si RNA in MSC to downregulate the Wnt/β-catenin pathway,in order to rescue the mitochondrial dysfunction.This finely designed nanoparticle"mitochondrial calcium calibrator"physiologically restored mitochondrial and MSC function and provided therapeutic benefits for periodontitis and osteoarthritis,which may further contribute to other mitochondrial quality control interventions.