Expression Of Laminin Receptor In Tooth Germ And Its Role In Odontoblast Differentiation

Background:Dental caries,dentin hypoplasia and other diseases are common oral diseases,which can cause dental defects,dentition defects and even dentition loss,affecting mastication,pronunciation and aesthetics.Therefore,the study of the formation process of dentin is of great significance for dentin regeneration.Dentin and enamel are secreted and mineralized by mature odontoblast and ameloblast respectively.During the development of tooth germ,with the differentiation of ameloblast and odontoblast,the morphology of the cell gradually changes from cuboidal to high columnar,the organelle is relocated,and the nucleus gradually moves away from the basement membrane.This functional and morphological asymmetry is called cell polarity.The secretory function of odontoblast and ameloblast depends on the establishment and maintenance of cell polarity.Previous studies have shown that the main components of the basement membrane,laminin and its receptor integrin β1 and laminin receptor 1（LAMR1）,play an important role in the establishment of cell polarity.There is also a basement membrane between ameloblasts and dental papilla during tooth development.However,there are few studies on the role of basement membrane in the polarity of ameloblasts and odontoblasts.Objective:In this study,we investigated the temporal and spatial expression of integrin β1and LAMR1,which are the main receptors of laminin in the basement membrane,during tooth development and their role in odontoblast differentiation.It provides a theoretical basis for the study of the polarity of ameloblasts and odontoblasts.Methods:The proximal and distal sections of mandibular first molars were prepared from mice at embryonic stage 13.5（embryonic day 13.5,E13.5）、E14.5、E16.5、E18.5 and5 days after birth（postnatal day 5,PN5）.The morphology of tooth germ was observed by HE staining and the expression pattern of integrin β1 and LAMR1 in mouse mandibular first molar germ was observed by immunohistochemical staining.The expression of integrin β1 during differentiation of human dental pulp stem cells（dental pulp stem cells,DPSCs）in vitro was analyzed by q RT-PCR and Western blot.The expression of integrin β1 in DPSCs was inhibited by si RNA gene silencing,DPSCs was induced by osteogenesis in vitro,and the expression of osteogenic related genes in DPSCs was detected by q RT-PCR.On the 7th day and 14 th day of induction,ALP staining were performed.On the 14 th day of induction,alizarin red staining were performed to analyze the role of integrin β1 in the differentiation of DPSCs in vitro.Results:HE staining showed that E13.5,E14.5,E16.5,E18.5 and PN5 corresponded to the bud stage,cap stage,early bell stage,late bell stage and crown development stage of mouse tooth germs.The results of immunohistochemical staining showed that integrin β1 and LAMR1 were expressed in both epithelium and dental papilla during mouse molar development,and their expression intensity gradually increased with the degree of cell differentiation.In PN5,integrin β1 and integrin β1 were strongly expressed in the secretory end of odontoblast and ameloblast.q RT-PCR and Western bolt analysis showed that the expression of integrin β1 increased gradually on the 0,3rd,7th,10 th and 14 th day of differentiation of DPSCs.The m RNA expression level of integrin β1 on the 10 th and 14 th day was significantly higher than that on the 0th day（P < 0.05 and P < 0.01 respectively）.The protein expression of integrin β1 on the10 th and 14 th day was significantly higher than that on the 0 day.Osteogenesis of DPSCs was induced in vitro when si RNA inhibited the expression of integrin β1 in DPSCs.q RT-PCR results showed that there was no significant difference in the expression of ALP and DSPP between the experimental group and the control group on the 7th day of induction,but on the 14 th day,the expression of RUNX2 in the experimental group was significantly lower than that in the control group（P < 0.01 and P < 0.05,respectively）.There was no significant difference in BMP2 between the two groups on the 7th day and 14 th day of induction.ALP staining showed that on the7 th day and 14 th day,the staining in the experimental group was significantly weaker than that in the control group,and alizarin red staining showed that the number of calcium nodules in the experimental group was significantly lower than that in the control group on the 14 th day of induction（P < 0.01）.Conclusion:（1）Integrin β1 and LAMR1 are expressed in odontoblast,ameloblast and basement membrane during mouse molar germ development.They may be involved in the signal transduction between cells and cell extracellular matrix,and play an important role in the differentiation and polarity formation of odontoblast and ameloblast.（2）Integrin β1 may play an important role in the differentiation of DPSCs.