The Role Of ACVR1 In Tooth Root Formation

Objectives:Tooth root plays an important role in bearing,transmitting and dispersing masticatory pressure.Short root anomaly,such as tooth root hypoplasia and absence of root,can lead to early tooth loss and affect pronunciation,aesthetics,masticatory function and patients’physical and psychological health severely.The root dentin,which is the main structure of the root,is formed by odontoblasts.Therefore,to explore the formation of the root dentin is of great significance to clarify the molecular mechanism of the root development and prevent early tooth loss due to short root.It has been reported that BMP signaling pathway and related transcription factors are involved in tooth root development,but the roles and mechanisms of BMP signaling in root dentin formation are not clear.In this study,we used the conditional knockout mice to explore the important roles of BMP signaling mediated by ACVR1in the differentiation of odontoblasts and the formation of root dentin.Methods:1.The Acvr1 gene was specifically knocked out in odontoblasts.The mandibles of the transgenic mice fed with normal diet with reporter gene were collected at postnatal day（PN）5.At the same time,the mandibles of the transgenic mice with reporter gene were collected at PN7,which were fed with doxycycline before birth and normal diet after birth.The expression of reporter gene was observed by fluorescence microscope,which indirectly reflected the localization of activated Cre recombinase.2.The mandibles of the control(Osterix-Cre;Acvr1 ^fx/+)mice and Acvr1conditional knockout（cKO）(Osterix-Cre;Acvr1 ^fx/-)mice at PN42 were collected.After fixation,the root lengths of the mandibular first molars were measured by micro-CT.The mandibles of the control and Acvr1 cKO mice at PN14/21/42 were collected.After fixation,decalcification,dehydration,and paraffin embedding were performed to obtain the sagittal sections of the mandibular first molars.The root lengths of the mandibular first molars were measured by HE staining.To detect the roles of ACVR1 in the formation of root dentin and the differentiation of odontoblasts,the protein levels of DSP,DMP1 and BSP in the odontoblasts in the root bifurcation of control and Acvr1 cKO were measured by immunohistochemical staining at PN14/42.The formation of collagen fibers was measured by Sirius red staining at PN42 to determine the effect of ACVR1 on collagen forming ability of odontoblasts.Results:1.The reporter gene was expressed in postnatal odontoblasts,suggesting that Osterix-Cre was activated in these cells.It also indirectly indicated that Acvr1 gene could be knocked out in postnatal odontoblasts.2.Conditional knockout of Acvr1 gene in odontoblasts resulted in short roots and abnormal dentin formation of the mandibular first molars in mice.Micro-CT and HE staining showed that the mice exhibited short mandibles,short roots and thin root dentin at PN14/21/42 after conditional knockout of Acvr1 gene.3.The odontogenic differentiation of odontoblasts is disrupted in Acvr1conditional knockout mice,and the secretion and formation of collagen were affected.Immunohistochemical staining showed that,compared with the control mice,the protein levels of DSP,DMP1 and BSP in the odontoblasts in the root bifurcation of Acvr1 cKO mice was not significantly different at PN14,however,the levels of DSP and DMP1 were decreased at PN42.The protein of DMP1 was not detectable in the cells embedded in the osteodentin.The level of BSP was not significantly different.Sirius red staining showed that the collagen fibers in the root dentin of Acvr1 cKO mice were yellow and green,and the arrangement was disordered,with the burr like protrusion protruded into the pulp cavity.These indicated that the tooth root dentin was mainly composed of small and immature collagen fibers in Acvr1 cKO mice.Conclusions:1.ACVR1 promoted odontogenic differentiation of odontoblasts.2.ACVR1 promoted the tooth root dentin formation.