Mechanistic Study Of Sema3A Regulation Of Osteoblasts In Peri-Implantitis Bone Homeostasis

Background and objectives:As one of the molecules that can regulate both osteoblasts and osteoclasts simultaneously,Semaphorin 3A(Sema3A)has been shown to play a decisive role in bone reconstruction and metabolism.However,its role and mechanism in the occurrence,development of peri-implantitis,and peri-implant bone homeostasis have not been fully elucidated.This study aims to observe the expression difference of Sema3A in peri-implantitis implants and healthy peri-implant gingival crevicular fluid,as well as its correlation with clinical indicators.At the same time,we investigate the effects of Sema3A on the proliferation,migration,and osteogenic differentiation of osteoblasts in the inflammatory microenvironment of bone homeostasis,as well as the relevant regulatory mechanisms.Methods:1.Enzyme-linked immunosorbent assay(ELISA)was used to measure the protein levels of Sema3A,bone formation markers procollagen Ⅰ N-terminal propeptide(PINP),and C-terminal telopeptide of type Ⅰ collagen(CTX-Ⅰ)in periimplantitis and healthy peri-implant gingival crevicular fluid.2.The correlations between the above proteins and clinical parameters such as probing depth(PD),modified sulcus bleeding index(mSBI),plaque index(PLI),and bone loss were analyzed.3.Mouse pre-osteoblast cell line(MC3T3-E1)was cultured and passaged.4.MC3T3-E1 cells were infected with a lentiviral vector overexpressing Sema3A.5.The effect of Sema3A overexpression on the proliferation of MC3T3-E1 cells treated with P.gingivalis lipopolysaccharide(LPS)was assessed using Cell Counting Kit-8(CCK8)assay and EDU assay.6.The effect of Sema3A overexpression on the migration of MC3T3-E1 cells treated with P.gingivalis LPS was evaluated using scratch assay and transwell chamber assay.7.The effect of Sema3A overexpression on the osteogenic differentiation of MC3T3-E1 cells treated with P.gingivalis LPS was assessed using Alizarin Red staining and alkaline phosphatase(ALP)staining.8.The effect of Sema3A overexpression on the mRNA and protein levels of some osteogenic factors in MC3T3-E1 cells treated with P.gingivalis LPS was determined using quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.9.Differential expression genes(DEGs)and related signaling pathways were identified and analyzed by RNA sequencing(RNA-seq)and functional analysis of screened genes.10.The effect of Sema3A overexpression on the PI3K/AKT signaling pathway in MC3T3-E1 cells treated with P.gingivalis LPS was assessed using Western blot.The impact of Sema3A overexpression on various phenotypes of MC3T3-E1 cells treated with P.gingivalis LPS after the addition of PI3K/AKT signaling pathway inhibitor P529 was evaluated using CCK8 assay,scratch assay,Alizarin Red staining,ALP staining,qRT-PCR,and Western blot.Results:1.Compared to healthy peri-implant gingival crevicular fluid,protein levels of Sema3A and PINP decreased while CTX-Ⅰ increased in peri-implantitis gingival crevicular fluid.2.Sema3A,PINP,and CTX-Ⅰ were highly correlated with PD and bone loss,while their correlation with mSBI and PLI was low.3.MC3T3-E1 cells were successfully cultured and exhibited good cell morphology with spindle-shaped cells and oval-shaped nuclei centrally located in the cells.After 4 days of culture,the cells reached a confluence of 70%-80% and could be used for subsequent experiments.4.Stable MC3T3-E1 cell lines overexpressing Sema3A were successfully constructed by infecting them with lentiviral vectors.5.CCK8 and EDU experiments showed that compared to the normal group,treatment with P.gingivalis LPS inhibited the proliferation of MC3T3-E1 cells,while overexpression of Sema3A upregulated the proliferation of MC3T3-E1 cells treated with P.gingivalis LPS.6.Scratch and transwell assays showed that compared to the normal group,treatment with P.gingivalis LPS inhibited the migration of MC3T3-E1 cells,while overexpression of Sema3A upregulated the migration of MC3T3-E1 cells treated with P.gingivalis LPS.7.Alizarin red and alkaline phosphatase(ALP)staining experiments showed that compared to the normal group,treatment with P.gingivalis LPS inhibited the osteogenic differentiation ability of MC3T3-E1 cells,while overexpression of Sema3A upregulated the osteogenic differentiation ability of MC3T3-E1 cells treated with P.gingivalis LPS.8.qRT-PCR and Western blot experiments showed that compared to the normal group,treatment with P.gingivalis LPS downregulated the BMP2,RUNX2,and ALP protein levels,as well as the RUNX2,ALP,and COL2 genes of MC3T3-E1 cells(p <0.05),while overexpression of Sema3A significantly upregulated the BMP2,RUNX2,and ALP protein levels(p < 0.05)and the RUNX2,ALP,and COL2 genes(p < 0.05)in MC3T3-E1 cells treated with P.gingivalis LPS.9.RNA-seq and functional analysis of screened genes showed that overexpression of Sema3A in P.gingivalis LPS-treated MC3T3-E1 cells led to 127 upregulated and 45 downregulated differentially expressed genes in the entire transcriptome,and the upregulation of Angpt1,Angpt2,and Nos3 genes suggested that overexpression of Sema3A may be closely related to the PI3K/AKT signaling pathway.10.Western blot analysis revealed that treatment with P.gingivalis LPS significantly downregulated p-AKT levels in MC3T3-E1 cells compared to the control group(p < 0.05),while overexpression of Sema3A significantly upregulated p-AKT levels in MC3T3-E1 cells treated with P.gingivalis LPS(p < 0.05).Additionally,the addition of the PI3K/AKT signaling pathway inhibitor P529 suppressed the ability of Sema3A overexpression to enhance proliferation,migration,and osteogenic differentiation of MC3T3-E1 cells treated with P.gingivalis LPS.Conclusion:Sema3A showed a trend of downregulation in peri-implantitis gingival crevicular fluid,accompanied by a decrease in PINP and an increase in CTX-1.These changes were closely related to probing depth and degree of bone loss.In in vitro experiments,overexpression of Sema3A could rescue the inhibitory effect of P.gingivalis LPS on the proliferation,migration,and osteogenic differentiation of osteoblasts in bone homeostasis.Furthermore,RNA-seq revealed that the effect of Sema3A in osteoblasts may be closely related to the PI3K/AKT signaling pathway.Finally,it was observed that overexpression of Sema3A could upregulate the p-AKT levels in osteoblasts treated with P.gingivalis LPS,and the PI3K/AKT signaling pathway inhibitor P529 could inhibit all the rescue effects of Sema3A overexpression on the proliferation,migration,and osteogenic differentiation of osteoblasts in bone homeostasis.We conclude that Sema3A can regulate peri-implantitis bone homeostasis by promoting the PI3K/AKT signaling pathway.