Study On The Ability Of Neural Crest Cells To Induce Dental Formation In Vitro

Lack of teeth has already been a difficult problem for people.It directly affects the physiological functions such as chewing,nutrition,digestion and absorption.At the same time,language function and facial image are affected by lack of teeth.At present,the main measure of repairing tooth damage is to use artificial prosthesis,but this is not a permanent solution method.The prosthesis has limited by the physiological function,short service life,high price,and poor sense of use,these shortcomings will always accompany people.Regeneration teeth are similar to natural teeth in morphology,structure and function,which can overcome the disadvantages of prosthesis.Therefore,regenerative teeth have become the latest concept of dental restoration.The development of teeth is a continuous and complex process.Neural crest cells play a very important role in this process.Tooth germ mesenchymal cells derived from neural crest cells can interact with tooth germ epithelial cells to develop into complete teeth.Therefore,neural crest cells can be used in the study of tooth regeneration.In this study,neural crest cells were isolated from pregnant mice at 9.5 days of gestation,and then differentiated them into tooth germ mesenchymal cells by means of neural crest cells specific medium induction.Identification of cultured neural crest cells by neural crest specific expression genes,the neural crest cells from different batches are recombined with the tooth germ epithelium to form in vitro recombinant tooth germs,and then recombinant tooth germs are transplanted into ICR mouse kidney capsule,the ability of the neural crest cells to form teeth is detected after 3 weeks.RNA sequencing technology was used to analyze the regulation pathway of neural crest cells into teeth.The experimental results showed that in the process of in vitro culture,a large number of neural crest cells migrated from embryonic neural tube.Microscopic observation of these migrated neural crest cells revealed that the main form is spindle type.Neural crest cells of p0-p5 generation were detected by qPCR,and it was found that neural crest specific genes Snail,hnk1,P75 and Pax3 were continuously expressed in the processes of in vitro culture.The result indicated that the neural crest cells cultured in vitro could always maintain the neural crest specific genes.Through recombinant transplantation,tissue staining and other experiments,it was found that the recombinant tooth germs formed by neural crest cells and tooth germ epithelium can develop into teeth in the kidney capsule of ICR mouse,the regenerated tooth has a relatively complete tooth structure,such as tooth enamel structure,dentin structure and tooth pulp structure,etc.Using RNA sequencing technology to analyze the metabolic pathways and biological processes involved in dental and non-tooth samples,according to the comments of KEGG database analysis,it was found that neural crest cells develop into teeth through the Wnt Signaling pathway and Hippo signaling pathway,the two pathways regulate the formation of teeth,according to the annotation analysis of the GO database,it was found that the reason why the neural crest cells could not develop into teeth was mainly because it differentiated into the nerve direction and did not differentiate into the tooth germ mesenchymal cells.In summary,the odontogenic potential of neural crest cells is confirmed by in vitro induction,gene testing,recombinant transplantation,tissue staining and other aspects.And then,RNA sequencing technology was used to further explore the relevant pathways and biological processes of neural crest cells in the processes of tooth development.In future experimental design,the key tooth-forming pathways and biological processes can be activated or inhibited to improve the odontogenic potential of neural crest cells.At the same time,these results provide a refenence data for the construction of regenerative teeth.