单细胞组学解析ACVR1在成牙本质细胞分化中的作用OA

Objective To investigate the impact of activin receptor type-1(ACVR1)on odontoblastic differentiation and dentin formation based on single-cell RNA sequencing.Methods A correlation analysis of ACVR1 with odontogenesis-related transcription factors was performed using single-cell data from healthy wild-type adult mouse inci-sors obtained from the Gene Expression Omnibus database(GSE146123).This experiment was approved by the Institu-tional Animal Care and Use Committee of the affiliated institution.Conditional knockout mice with ACVR1 deleted in the dental mesenchyme were generated using the Cre-LoxP system(experimental group:Osterix-Cre;ACVR1fx/-,control group:Osterix-Cre;ACVR1fx/+).Dental pulp tissue was extracted from the incisors of 3-week-old mice to prepare single-cell suspensions for 10×Genomics single-cell transcriptome sequencing.Quality control,normalization,dimensionality reduction,and Harmony batch effect correction were performed using the Seurat pipeline,followed by UMAP dimen-sionality reduction visualization and cell subpopulation identification.Monocle was used for pseudotime analysis to infer cell differentiation trajectories,and changes in transcription factors positively correlated with ACVR1 were ana-lyzed at the single-cell data level.Immunofluorescence staining was employed to validate the localization and expres-sion changes of the key transcription factor Osterix and the odontoblast-specific protein DSPP.Results Sp7/Osterix is widely expressed in the incisor mesenchyme of mice(GSE146123).Using Osterix-Cre,ACVR1 was effectively knocked out in the mesenchyme.Gene correlation analysis between ACVR1 and key odontogenic genes showed positive correla-tions between ACVR1 and genes such as Msx1,Msx2,and Sp7.Single-cell atlases of incisor dental pulp tissue from"Osterix-Cre;ACVR1fx/-"and"Osterix-Cre;ACVR1fx/+"mice were then constructed.The most significant increase in the proportion of pre-odontoblasts was in the experimental group,along with downregulated expression of key genes re-lated to odontoblastic differentiation and polarization in this subpopulation.Differential gene enrichment analysis across all mesenchymal subpopulations indicated downregulated expression of most genes associated with dentin formation.Fi-nally,as observed through single-cell differential gene analysis,pseudotime expression analysis,and immunofluores-cence validation,the expression of Sp7 and DSPP were significantly downregulated in the experimental group.Conclu-sion The ACVR1 gene may promote odontoblastic differentiation and subsequently affect dentin formation through transcription factors such as Sp7,Msx1,and Msx2.