Dicalcium Silicate Modulates Osteogenesis In Bone Marrow Mesenchymal Stem Cells Via The CircRNA₁983

BackgroundDicalcium silicate?C₂S?is a biologically active CaO-SiO₂ calcium-silicon based material,which has broad development potential in bone tissue engineering applications.Previous studies have shown that,compared with hydroxyapatite,dicalcium silicate have a lower pro-inflammatory effect and a stronger pro-mineralization effect.At present,research on it is mainly concentrated in the fields of powder synthesis,coating,bulk ceramics,and so on,and its molecular mechanism in the process of exerting biological functions is rarely reported.The regulatory role of messenger RNA?mRNA?in osteogenic differentiation of mesenchymal stem cells?MSCs?has been widely studied,but whether dicalcium silicate works through well-defined osteogenic pathways remains unknown.At the same time,with the development of high-throughput sequencing technology,many scholars have found that Circular RNA?circRNA?can promote osteogenic differentiation of bone marrow mesenchymal stem cells and play a role through the ceRNA?circRNA-miRNA-mRNA?interaction network.However,the molecular mechanism of C₂S-mediated bone regeneration is not fully understood,and the effect of C₂S on circRNA expression in MSCs and the role of circRNA in bone regeneration remain unknown.In the Pubmed database search,we didn't find the keywords for"Dicalcium Silicate and CircRNA".Therefore,it is of great significance to study the role and mechanism of biomaterials in circRNA-mediated bone defect healing.ObjectiveIn this study,we aim to clarify the osteogenic ability of dicalcium silicate particles,elucidate the differential expression of circRNA and mRNA in C₂S-treated MSCs,and the role of circ?1983 in C₂S-mediated bone regeneration,and its mechanism.Material and Methods1.Dicalcium silicate is added to the osteogenesis induction process of mouse bone marrow mesenchymal stem cells.In vitro induction of dicalcium silicate is detected by alkaline phosphatase activity detection,alizarin red staining test,and expression of osteogenesis-related genes.Osteogenic function,and through the rat skull defect model,to test whether dicalcium silicate can induce osteogenesis in vivo.2.High-throughput sequencing of mice bone marrow-derived MSC?mBMSC?in normal culture,mBMSC in osteogenic induction culture,and mBMSC in osteogenic induction culture with dicalcium silicate.The mRNA and circRNA expression profiles were constructed,and differentially expressed mRNAs and circRNAs were screened.The host genes of differentially expressed mRNAs and circRNAs were analyzed by GO analysis and KEGG pathway enrichment.Correlation analysis was performed on differentially expressed mRNAs and circRNAs,and multiple target gene prediction databases were used to predict miRNAs that might interact with them to construct a circRNA-miRNA-mRNA regulatory network.3.The prediction upstream of differential gene Gas7,circ?1983 was selected for verification,q-PCR method was used to verify the relative expression level to confirm the reliability of sequencing results,specific back-to-back primers were designed and primer specificity was verified by Sanger sequencing,and RNase R tolerance Sex experiment to verify the authenticity of circ?1983.Construction of circ?1983interference vector,shRNA transfection of mBMSC to interfere with circ?1983expression,and explore the effect of circ?1983 on dicalcium silicate promotes mBMSC osteogenesis to differentiation phenotype;finally,by detecting the expression level of target miRNA,to determine the possible targets of circ?1983,and preliminary exploration of the mechanism of circ?1983 in the process of dicalcium silicate promoting bone regeneration.ResultsC₂S enhanced osteogenic differentiation of mBMSC and bone defect healing.The C₂S group had stronger expression levels of alkaline phosphatase and more obvious ability to promote cell mineralization in vitro.Compared with the control group,there were 3474 circRNAs and 6575 mRNAs differentially expressed in C₂S treated mBMSC.1811 mRNA-miRNA-circRNA interaction networks were successfully constructed.The differentially expressed mRNA,circRNA,and ceRNA interaction networks are related to osteogenesis-related pathways such as PI3K-Akt and MAPK.We observed the association between circ?1983,Gas7 and Runx2expression in C₂S-treated mBMSC,and proposed that the ceRNA regulatory network circ?1983–miR-6931–Gas7 can enhance Runx2 expression and promote osteogenesis,successfully designed specific primers for circ?1983,and clarified the stability of circ?1983.circ?1983 knockdown down-regulated Gas7 expression and inhibited C₂S-induced mBMSC osteogenic differentiation.ConclusionStudies have shown that C₂S has a strong ability to promote bone regeneration in vivo and in vitro.In the process of C₂S promoting osteogenesis,it can cause differential expression of mRNA and circRNA,and may participate in biological processes related to osteogenesis through MAPK,PI3K-AKT and other signaling pathways.Interfering with circ?1983 expression can influence the osteogenic differentiation of mBMSC induced by C₂S.C₂S may promote osteogenesis through the circ?1983-miR-6931-Gas7 interaction network.