The Role Of ACVR1 In Promoting Odontogenic Differentiation Of Dental Pulp Cells Via AUTOTAXIN/LPA Axis

Objective: Dental tissue defects such as caries affect the mastication,digestion and absorption functions of patients.Large area of tooth tissue defects can lead to early tooth loss,and even affect the development of maxillofacial and mental health of patients.The treatment strategy of tooth tissue defect is mainly to restore the morphology of tooth tissue and promote the formation of restorative dentin.However,the restorative dentin formed at present often lacks the structure of dentin tubules,and is bone-like dentin.This dentin cannot play its normal physiological functions such as external stimulation and mechanical conduction.Therefore,exploring the formation process and molecular mechanism of dentin under physiological conditions will help to reveal the key factors of dentin formation,provide new targets for promoting the formation of restorative dentin with physiological functions and even dentin regeneration,and have important potential significance for the treatment strategy of dental tissue defects in clinical practice.Although it has been confirmed that BMP signal plays an important role in odontoblast differentiation and dentin formation,because BMP signal pathway contains a variety of ligands and receptors,its mode of action and time of action play different roles in different stages of tooth development,and its specific mechanism is still unclear.In this study,type I BMP receptor activator type 1(ACVR1)was selected as the research target to explore the role and molecular mechanism of BMP signal pathway mediated by ACVR1 in odontoblast differentiation and dentin formation,and provide theoretical basis for clarifying the molecular mechanism of BMP signal promoting dentin formation.The formation of dentin is a complex process in which multiple cell types,multiple genes and multiple signal pathways work together in a coordinated or antagonistic manner.The dental pulp cells themselves are composed of a variety of heterogeneous cell subsets.In this paper,the mouse incisor pulp,in which cells at different stages of differentiation can exist at the same time,was used as the research object.The single-cell sequencing technology was used to screen and classify the various cell types involved in dentin formation one by one,so as to clarify the key role of these cells in the ACVR1-mediated BMP signal pathway in promoting odontoblastic differentiation and dentin formation.Lysophosphatidic acid(LPA)is a biologically active phospholipid,which is produced by the hydrolysis of extracellular lysophosphatidylcholine(LPC)by the protein encoded by the Enpp2 gene,Autotoxin(ATX).Therefore,we will explore the ATX/LPA axis as a whole.LPA can promote the migration and adhesion of human dental pulp stem cells,and participate in enamel formation through receptor LPA6.However,it is still unclear whether ATX/LPA axis can promote dentin formation and whether ATX/LPA axis can save the odontogenic differentiation ability of ACVR1 gene silenced h DPSCs.This experiment aims to clarify that ACVR1-mediated BMP signal can promote odontoblast differentiation and dentin formation through ATX/LPA axis.Methods: 1.HE staining was used to detect the odontoblast differentiation and dentin formation in the mandibular incisors of control and Acvr1 conditional knockout mice.Immunofluorescence staining was used to detect the distribution and proportion of Ki67 positive proliferative cells in the mandibular incisors of control and Acvr1 conditional knockout mice.2.The major cell subpopulations and marker genes of all cells and Lac Z positive cells were separately analyzed by single cell sequencing technology in dental pulp tissue of Osterix-Cre;Acvr1 fx/+(control)and Osterix-Cre;Acvr1 fx/+(c KO)mice.The expression and distribution of Enpp2 and Cldn10 genes in dental pulp of control and c KO mice was detected by q PCR and RNAScope in situ hybridization.The differential genes between control and c KO mice were screened and analyzed by GO and KEGG enrichment.3.HE staining was used to detect the odontoblast differentiation and dentin formation of the mandibular incisors in the control group and PF8380 group.Human dental pulp stem cells(h DPSCs)were identified by flow cytometry,alizarin red staining,oil red O staining and alcian blue staining.The type and quantity of LPA receptor expressed in h DPSCs after 0/7/14 days of osteogenesis induction were detected by q PCR.CCK8 was used to detect the survival rate of h DPSCs at different time after adding different concentrations of LPA.The effect of LPA on odontogenic differentiation of h DPSCs was detected by alkaline phosphatase(ALP)staining.The transfection efficiency of si RNA was observed by fluorescence microscopy.The efficiency of ACVR1 gene silencing was detected by q PCR.The odontogenic differentiation ability of h DPSCs after ACVR1 gene silencing was detected by ALP staining,Western blot,q PCR and alizarin red staining.4.Statistical analysis: all in vitro experiments were repeated for 3 times,and in vivo animal experiments were repeated for at least 2 times.The experimental results were expressed as mean ± standard deviation.The two groups of data in accordance with normal distribution were statistically analyzed by Student’s t test.p<0.05 indicates statistical difference.Results:.1.HE staining results showed that compared with the control group,the differentiation of odontoblasts and the formation of dentin were abnormal in the Acvr1 conditional knockout mice.Immunofluorescence showed that compared with control mice,the proportion of Ki67 positive proliferative cells in the mandibular incisors of Gli1-Cre ERT2;Acvr1 fx/fx mice increased significantly,although there is no significant difference in c KO mice.2.The single-cell sequencing results showed that the dental pulp tissue(including dental epithelium)of control and c KO mice mainly consisted of 8 cell types and 24 clusters,the cells expressing Lac Z gene in the dental pulp tissue of control and c KO mice mainly included six subgroups,and the proportion of both Enpp2 and Cldn10 positive cells decreased.q PCR results showed that compared with control group,the expression of Enpp2 gene and Cldn10 gene in dental pulp tissue of c KO group decreased significantly(p=0.08).The results of RNAScope in situ hybridization showed that the double positive cells of Enpp2 and Cldn10 were located in the lower layer of odontoblasts.Compared with the control group,the double positive cells of Enpp2 and Cldn10 in the dental pulp of c KO group mice were significantly reduced.The results of GO enrichment analysis showed that compared with the control group,the up-regulated genes in the c KO group were related to the negative regulation of phosphate metabolism and ion transport,while the down-regulated genes in the c KO group were related to extracellular matrix and mineralization.The results of KEGG enrichment analysis showed that the up-regulated genes in c KO group were related to MAPK signal pathway,while the down-regulated genes in c KO group were related to tight junction,cytoskeleton and PI3K-Akt signal pathway.The GO and KEGG enrichment analysis of down-regulated genes between Enpp2 and Cldn10 double positive cells showed that down-regulated genes in c KO group were related to ossification,odontogenesis and PI3K-Akt signal pathway.3.The HE staining results showed that compared with the control group,the odontoblasts of the mandibular incisors of mice in PF8380 groups became shorter and the dentin became thinner in different degrees.The results of flow cytometry showed that h DPSCs expressed mesenchymal stem cell molecular markers CD90 and CD105 positively,while negative expression of hematopoietic stem cell molecular markers CD34 and CD45.The results of alizarin red staining,oil red O staining and alcian blue staining showed that h DPSCs had the ability of osteogenic differentiation,adipogenic differentiation and chondrogenic differentiation.The results of q PCR showed that with the increase of odontogenic induction days,the expression of the other five LPA receptors increased except for LPA3.CCK8 results showed that the selected concentration of LPA had good biocompatibility,and the cell survival rate was not significantly affected.ALP staining showed that the alkaline phosphatase activity of the group with osteogenic induction fluid was significantly higher than that of the group without osteogenic induction fluid;At the same time,compared with the control group that only added osteogenic induction fluid,the alkaline phosphatase activity of LPA treatment group was stronger.Under fluorescence microscope,FAM-si RNA was successfully transferred into most cells.q PCR results showed that the silencing efficiency of ACVR1-Home-1218(si ACVR1)was higher and more stable.The ALP staining results showed that the alkaline phosphatase activity of the osteoinductive fluid group was higher than that of the group without osteoinductive fluid;Compared with the control group that only added osteogenic induction fluid,the alkaline phosphatase activity of ACVR1-Home-804 and ACVR1-Home-1218 transfected groups decreased.Western blot showed that the expression of DSPP and ATX protein in the group with osteoinductive fluid was higher than that in the group without osteoinductive fluid;compared with the control group with only osteogenic induction fluid,the expression of DSPP and ATX protein in the group with transfection reagent(Trans)and meaningless si RNA sequence(NC)both increased;Compared with the NC group,the expression of DSPP and ATX protein in the group with si ACVR1 decreased.The results of q PCR showed that the level of gene expression of ACVR1 was significantly decreased after silencing whether LPA was added or not;compared with the NC group,the expression of RUNX2 and ALP genes related to odontogenic differentiation of si ACVR1 group was reduced,but the expression of these two genes was partially rescured after adding LPA.Alizarin red staining showed that the calcium nodules in the osteoinductive fluid group were more than those in the group without osteoinductive fluid;compared with NC group under osteogenic induction,the number of calcium nodules was significantly reduced by silencing ACVR1 gene under osteogenic induction.However,the number of calcium nodules increased after adding LPA,which was similar to NC group.Conclusion: BMP signal mediated by ACVR1 is essential for the odontogenic differentiation of mesenchymal cells;BMP signal mediated by ACVR1 can maintain the number of Enpp2 and Cldn10 double positive cells under the odontoblastic layer,which may regulate the differentiation of odontoblasts;Both ATX and LPA can promote the differentiation of odontoblasts and the formation of dentin;ATX and LPA are downstream of ACVR1;ACVR1 promotes odontogenic differentiation of human dental pulp stem cells through ATX/LPA axis.