ACVR1 Promotes Dentin Formation Through Polarization Of Odontoblasts In Vivo

Objective:Diseases,such as dentinogenesis imperfecta,often cause defects of dentin formation and loss of dentin structure,further leading to severe attrition and even tooth loss,which in turn affects the patients’chewing,pronunciation,and aesthetics.Dentin is the main structure of teeth and is formed by mineralized matrix secreted by odontoblasts.Therefore,the study of odontoblast differentiation and its regulatory factors is of great significance for the understanding of the molecular mechanism of dentin formation,and has certain practical significance for the further regeneration of dentin.The differentiation of odontoblast and dentin formation are regulated by many signals,among which the Bone morphogenetic protein（BMP）signaling pathway plays an important role in the early stage of odontoblast differentiation and dental development.Polarization of odontoblasts is known to be a key step in the differentiation of odontoblasts,but there are few studies on the polarity of odontoblasts and its influencing factors.This experiment aims to investigate the mechanisms of BMP receptor activin A receptor type 1（ACVR1）on the regulation of the polarization of odontoblasts and formation of dentin in mice.The results will provide experimental basis for promoting dentine regeneration by regulating BMP signaling.Methods:The Acvr1 gene in mouse dental mesenchymal cells was knocked out by activating Cre recombinase with Osterix as the promoter by Cre-loxP system.To generate Acvr1 conditional knockout mice,mice homozygous for the conditional allele(Acvr1 ^fx/fx)were bred with mice heterozygous for the null allele carrying Osterix-Cre(Acvr1^+/-;Osterix-Cre（+）/（-）).And the conditional knockout（cKO）mouse genotype was Osterix-Cre（+）/（-）;Acvr1 ^fx/-,while the genotype of control（control,Cont.）mice is Osterix-Cre（+）/（-）;Acvr1 ^fx/+.The knockout group and control group specimens were collected at postnatal day 21（PN21）,and the morphologies of odontoblasts and dentin were observed by Hematoxylin-eosin（H&E）staining.Immunofluorescence（IF）staining was used to detect the localizations of mouse tight junction marker zonula occludens 1（ZO-1）,apical polarity marker protein kinase C zeta（PKCζ）,and Golgi marker Golgi Matrix Protein 130（GM130）.The arrangement and maturity of dentin collagen was assessed by Picrosirius red staining.Results:H&E staining showed that the boundary between mineralized dentin and predentin,as well as the boundary between predentin and odontoblast layer were smooth,while the boundaries of cKO group were zigzag.The dentin tubules of control group were arranged regularly at the cusps,and the odontoblasts were columnar and formed a palisading pattern,while the dentin tubules of cKO group at the cusps disappeared,and odontoblasts were embedded in the dentin matrix,forming osteodentin,and the high columnar morphology of the odontoblasts was lost.IF staining showed that ZO-1 and PKCζwere orderly expressed in the apical regions of odontoblasts in the control group,while ZO-1 and PKCζwere localized in the basal or basolateral regions of odontoblasts after Acvr1 gene was knocked out.GM130 was localized apical to the nucleus in the control group,while in the cKO group most Golgi apparatus were located apical to the nucleus and others were located at the basolateral regions of the nucleus.Additionally,after Picrosirius red staining,the collagen fibers in the control group were bright red and the maturity of collagen was high under polarized light,while in the cKO group,the collagen fibers were sparse and irregular,and the tissues were yellow-green with low maturity.Conclusion:ACVR1 in dental mesenchyme induces the formation of dentin by promoting the polarity of odontoblasts in mice.