Research On Mechanism Of Bioactive Glass-regulated Extracellular Vesicles Inhibit Osteoporotic Bone Loss

Bone tissue is an important part of the human body and has irreplaceable physiological functions.The growth of the vascular network in the skeletal system is regulated by signals released by bone cells such as osteoclasts and osteoblasts.Effective communication between these cells is essential for maintaining bony homeostasis,and extracellular vesicles are important intercellular communication mediators,rich in bioactive molecules that transmit genetic information and mediate cell-to-cell communication in vivo.In recent years,longstranded non-coding RNA(lncRNA)has become a key mediator in regulating paracrine signaling and cell crosstalk.lncRNA constitute cargo of extracellular vesicles that can shuttle from the donor to the recipient cell.lncRNA belongs to a class of RNAs that do not have coding ability,which has received a lot of attention in the past few years and has become an important player in biological regulation.More and more lncRNA are involved in controlling the function of important genes and regulating the fate of cells.For example,lncRNA is involved in physiological and pathophysiological processes of the endocrine system,reproductive system,metabolic system,immune system,nervous system,cardiovascular system.In this project,micro-nano bioactive glass is used to change the physiological environment of cells.By harnessing the ion release of bioactive glass in the humoral environment,as well as the characteristics of gene activation of cells.To investigate the regulatory effect of micro-nano bioactive glass on the secretion of bone marrow mesenchymal stem cells or macrophages,which promote osteogenic differentiation or inhibit osteoclastic differentiation.The lncRNA or cytokines in heterogeneous extracellular vesicles that promote osteogenic differentiation or inhibit osteoclastic differentiation were analyzed,and the differentially expressed lncRNA in heterogeneous extracellular vesicles was explored to promote osteogenic differentiation or inhibit osteoclastic differentiation and the regulatory effect of cytokines loaded on the immune microenvironment.In addition to biological functions,extracellular vesicles also have the advantages of small size,stable structure and low toxicity,and have shown broad application prospects in the field of nanomedicine.This topic discusses the effect and mechanism of heterogeneous extracellular vesicles regulated by micro-nano bioactive glass on osteogenic differentiation and osteoclastic differentiation,and further elaborates the new mechanism of micro-nano bioactive glass to promote osteogenic differentiation and inhibit osteoclastic differentiation.The biosafety and pharmacokinetic properties of heterogeneous extracellular vesicles were evaluated,which provided a theoretical basis and basis for the further development of nanomedicines for the treatment of osteoporosis.The main research work and conclusions of this paper are as follows:(1)Micro-nano bioactive glass nanoparticles(BGN)were prepared by sol-gel method combined with organic template method.The prepared BGN was used to induce the differentiation of mouse bone marrow mesenchymal stem cells(m BMSCs)to osteoblasts,and the effect of BGN on the expression difference of related lncRNA during osteogenic differentiation of m BMSCs was investigated by high-throughput sequencing.Biologics data analysis and transcriptome sequencing were used to predict and verify the biological function of lncRNA associated with osteogenic differentiation.The molecular biological mechanism by which this lncRNA affects osteogenic differentiation was verified at the cellular and animal levels.A new mechanism of BGN in promoting osteogenic differentiation was revealed.The results showed that Gm16263 is a new lncRNA associated with BGN-induced osteogenic differentiation of m BMSCs.In vitro experiments have shown that lncRNA Gm16263 is overexpressed in m BMSCs,which promotes the differentiation of m BMSCs into osteoblasts by increasing the expression of the downstream protein Taok3.In vivo experiments showed that EVs-Gm16263 had a better therapeutic effect than EVs-NC in the treatment of osteoporosis mice,and the expression of Taok3 protein in bone tissue was correspondingly increased.It shows that Gm16263 in vivo can also promote osteogenic differentiation and promote bone regeneration.It was revealed that the mechanism of BGN promoting osteogenic differentiation of m BMSCs was revealed by promoting the expression of Gm16263,which in turn promoted the expression of Taok3 protein,thereby promoting osteogenic differentiation of cells.(2)BGN-induced extracellular vesicles(BGN+BMSC-EVs)derived from bone marrow mesenchymal stem cells were successfully prepared and isolated by co-culture of BGN and m BMSCs,and the effect of BGN+BMSC-EVs on osteoclastic differentiation of osteoclast precursor cells was investigated.Combined with the sequencing data of BGN-induced osteogenic differentiation of m BMSCs,the expression of inclusions in BGN+BMSC-EVs was explored,and the mechanism of BGN+BMSC-EVs inhibiting osteoclastic differentiation in osteoclast precursor cells was explored.The biological function of BGN+BMSC-EVs was verified in osteoporosis mice,and their potential as a potential nanomedicine for the treatment of osteoporosis was evaluated,revealing a new mechanism of BGN in promoting osteoblastic differentiation or inhibiting osteoclast differentiation.The results showed that BGN+BMSCEVs had a more significant ability to inhibit osteoclast differentiation in in vitro experiments.BGN inhibits nuclear transfer of NFATc1 by activating the expression of lncRNA-Nron in BGN+BMSC-EVs,thereby inhibiting osteoclast differentiation.In vivo experiments have shown that BGN+BMSC-EVs have better therapeutic effect than BMSC-EVs in the treatment of osteoporosis mice,and can be used as a potential osteoporosis treatment drug.It was revealed that BGN can not only promote osteogenic differentiation by direct action with m BMSCs,but also induce m BMSCs to secrete extracellular vesicles,inhibiting the differentiation and activity of osteoclasts by cellular communication.(3)Micro-nano bioactive glass nanoparticles(SrBGN)doped with strontium were prepared by sol-gel method combined with organic template method.SrBGN+Mφ-EVs were successfully prepared and isolated by co-culture with bone marrow-derived macrophages(BMDMs),and RNAK on the surface of BMDMs and SrBGN+Mφ-EVs was determined.According to the research background of RANK-RANKL blocking therapy,the performance of SrBGN+Mφ-EVs combined with RANKL binding was studied,and the results showed that SrBGN could regulate the expression of more RANK protein on the surface of BMDMs,and make the surface of SrBGN+Mφ-EVs secreted have more RANK carry.RANK on the surface of SrBGN+Mφ-EVs is biologically active and can competitively bind to RANKL,block the RANK-RANKL signaling pathway,and inhibit the activity of osteoclasts.The RANK on the surface of SrBGN+Mφ-EVs per unit mass is higher than that of Mφ-EVs,which has higher binding efficiency for RANKL,and SrBGN+Mφ-EVs has a stronger effect on inhibiting osteoclastic differentiation.(4)The effect of SrBGN on bone marrow-derived macrophages was further explored,the active components of SrBGN+Mφ-EVs secreted by SrBGN+Mφ-EVs were investigated,and the effects of SrBGN+Mφ-EVs on the regulatory ability of Treg/Th17 cells,as well as the effects of regulation on osteogenic differentiation of bone marrow mesenchymal stem cells and osteoclasts.The biological function of SrBGN+Mφ-EVs was verified in osteoporosis mice.The results showed that SrBGN could induce BMDMs to polarize to M2 macrophages to a certain extent.The heterogeneous extracellular vesicles SrBGN+Mφ-EVs that interact with SrBGNs and BMDMs carry the bioactive substances of parent cells,namely anti-inflammatory factors TGF-β,IL-1Ra and IL-10,and the bioactive ions SrBGN and Si.SrBGN+Mφ-EVs can regulate Treg/Th17 cell balance in vitro,and promote osteogenic differentiation of bone marrow mesenchymal stem cells and osteoclasts.In vivo experiments showed that SrBGN+Mφ-EVs alleviated bone loss in osteoporotic mice by regulating the balance of Treg/Th17 in the spleen and peripheral blood of osteoporotic mice.SrBGN+Mφ-EVs can be used as a potential osteoporosis treatment.It was revealed that SrBGN can not only participate in the immune regulation of osteogenic differentiation with BMDMs through direct action,but also induce BMDMs to secrete extracellular vesicles,regulate Treg/Th17 balance in the form of cellular communication,improve the immune microenvironment,and alleviate bone loss.In summary,on the one hand,micro-nano bioactive glass can directly act on mesenchymal stem cells to promote the expression of lncRNA Gm16263 in mesenchymal stem cells,and then promote the expression of Taok3,thereby promoting osteogenic differentiation.It can also directly affect macrophages,regulate the differentiation of macrophages to M2,and improve the immune microenvironment.On the other hand,by stimulating mesenchymal stem cells to secrete extracellular vesicles,it increases the expression of lncRNA Nron in extracellular vesicles,and inhibits the nuclear transfer of Nfatc1 in osteoclast precursor cells,thereby inhibiting osteoclast differentiation and alleviating bone loss.It can also stimulate the secretion of extracellular vesicles by bone marrow-derived macrophages,produce inhibitors that can block the RANK-RANKL pathway and regulate the balance of Treg/Th17 cells,improve the immune microenvironment,promote osteogenic differentiation,inhibit osteoclastic differentiation,and alleviate bone loss.This paper elucidates the new mechanism of micro-nano bioactive glass in bone regeneration from a new perspective.In the process of research,the evaluation of the biological function of extracellular vesicles and the evaluation of biological safety and pharmacokinetic characteristics provide a theoretical basis and basis for the further development of nanomedicines for the treatment of osteoporosis.