The Spatio-temporal Expression Of P75NTR And RUNX2 During Mandibular First Molar Tooth Germ Early Development In Rat

ObjectiveTissue-engineered tooth research is undoubtedly one of the important research focusing in the oral medicine in recent years,but the precise mechanism of the tooth development is not yet clear,which restricts the development of tissue engineered tooth.And the cell reunion technology that simulates the tooth development between embryonic cells and mesenchymal epithelial function theory provides a new way for the development of tissue engineered tooth.As the primogenitor of dental stem cells,the cranial neural crest ectomesenchymal stem cell(EMSCs)are the ideal seed cells to obtain regeneration teeth by reunited cell technology,which are considered to be an important cell source of most parts of teeth organizations development except the enamel.p75 neurotrophin receptor(p75NTR)is a kind of neurotrophic factor receptor with low affinity.It is considered as a sort of cranial neural crest classic marker,one of the major membrane receptor and may be involved in tooth formation.Studies have found that p75 NTR in the non-dental origin cells may participate in the mineralization,and is related to Runt-related transcription factor 2(RUNX2).The mineralization of dental origin cells may be different from other cells,whether p75 NTR is involved in mineralization in dental origin cells and the relationship with RUNX2 are less reported.We will observe the expressions of p75 NTR and RUNX2 of SD rats mandibular first molar in different developing stages,discuss its effect of developing and mineralization on mandibular first molar after the birth of SD rats and set forth its mechanism.Therefor,it is helpful to provide the experimental basis for revealing molecular biology mechanism of toothdevelopment,and search for a good seed cells for tissue engineered tooth.Methods1 Haematoxylin-eosin(HE)staining to observe the morphology of mandibular first molar tooth germ of different embryonic age of rat.2 percent sodium pentobarbital(40mg/kg)were injected into the abdominal cavity of the embryo 13.5d,14.5d,15.5d,16.5d,18.5d and postnatal 0.5d spraguedawley(SD)rats.After waiting for about 5min,the embryo was taken out by abdominal surgery,and then fixed in the 4% paraformaldehyde for 24 h,embedding,slicing the tissue in 6 um thickness by coronal plane,HE staining,in the last observe under the microscope and taking pictures.2 The spatio-temporal expression of p75 NTR during mandibular first molar tooth germ early development in rat.Rewarming previous E13.5d?E14.5d?E15.5d?E16.5d?E18.5d?P0.5d frozen section for 30 min,in acetone for 10 minutes,then wash in PBS,immunostaining were performed using rabbit monoclonal p75NTR(1:1500,abcam,Cambridge,UK)by DAB Detection Kit Streptavidin-Biotin)(ZSGB,Beijing,CHN)according to the manufacturer's protocols,Hematoxylin staining,in the last observe under the microscope and taking pictures.3 The spatio-temporal expression of RUNX2 during mandibular first molar tooth germ early development in rat.Rewarming previous E13.5d?E14.5d?E15.5d?E16.5d?E18.5d?P0.5d frozen section for 30 min,in acetone for 10 minutes,then wash in PBS,immunostaining were performed using rabbit monoclonal RUXN2(1:2000,abcam,Cambridge,UK)by DAB Detection Kit Streptavidin-Biotin)(ZSGB,Beijing,CHN)according to the manufacturer's protocols,Hematoxylin staining,in the last observe under the microscope and taking pictures.4 Mineralized induced P0.5d EMSCs in vitro4.1 Culture and identify the ectomesenchymal stem cells of P0.5d SD rats in the vitro.2 percent sodium pentobarbital(40 mg/kg)were injected into the abdominal cavity of the postnatal 0.5d SD rats.After waiting for about 5 min,the embryo was taken out by abdominal surgery,Looking for the mandibular first molar germs,After pancreatic enzyme digestion,centrif?gal and filtering by the 100 mesh stainless steel filter of the tissue,the tissue were respectively treated with culture medium of fetal bovine serum,then put into 37?,5%CO2 incubation cultivation box.The biology identification of P0.5d EMSCs were detected respectively by the cell morphology,cell immunofluorescence and flow cytometry technique to detect cell surface antigen.4.2 Mineralized induced P0.5d EMSCs in vitro and analysis.The P0.5d ectomesenchymal stem cells were mineralized induction for 3,7,14,21 days In vitro,each groups collected proteins.To detect the expression of proteins of p75 NTR and RUNX2.Statistical methods: Using SPSS Statistics 22.0 statistical analysis software,the data be shown by mean ± standard deviation,the repeated measures engineered variance analysis was performed to compare differences for individual time points.Multiple comparison between the groups choose paired LSD,datas are compaired by using pearson correlation analysis.P<0.05 for the difference was statistically significant.Results1.E13.5d growed into the grey shape of tooth germ,that the tip of dental plate enlarged,and hyperplasia epithelial cells form ovoid or round epithelium buds,shaped like a bud.The tooth germ epithelial bud of E14.5 d rat continue to grow?increase into epithelial basel parts and inward concave,which shaped like hat,and cover the accumulation of embryo mesenchymal cells,which called cap stage of initial morphogenesis.The tooth germ of E15.5d rat was in end of the cap stage,the enamel orange is divided into three layers: the outer enamal epithelium,stellate reticulum and inner enamal epithelium.The cell clusters of the bottom of the enamal orange was called dental papilla,which will form dentin and dental pulp;and the embryo mesenchymal cells circumvolutioed by the enamel organ and dental papil was called gums,which form teeth support groups.The tooth germ of E16.5d rat was bell stage of initial morphogenesis,The tooth germ of E18.5d rat was in period of bell stage.The epithelium sag to deepen,and peripheral parts continue to grow.At this time enamal organ enter into the mature period,divided into four layer: outer enamal epithelium,stellate reticulum,stratum intermedium and inner enamal epithelium.At P0.5d(The bell stage of differentiation)The inner enamel epithelium cells near the dental papilla,differentiation for preameloblast,part of the dental papilla cells adjacent the basement membrane began to differentiate into odontoblasts,the other part differentiation with odontoblasts secrete function.On the tip of the dental papilla,part of odontoblasts secrete for predentin matrix.2.We investigated p75 NTR expressed in interstitial tissue near the enamel organ at the bud stage(E13.5d);in inner enamal epithelium,enamel knot,stellate reticulum,dental papilla,dental sac at the cap stage(E14.5 d and E15.5d);in inner enamal epithelium,enamel knot,stellate reticulum,stratum intermedium,dental papilla,dental sac at the bell stage(E16.5d,E18.5d and P0.5d).3.RUNX2 detected in interstitial tissue under the enamel organ at the bud stage and cap stage of initial morphogenesis(E13.5 d and E14.5 d);in inner enamal epithelium,enamel knot,stellate reticulum,dental papilla,dental sac at the end of the cap stage(E15.5 d);in inner enamal epithelium,enamel knot,stellate reticulum,stratum intermedium,dental papilla,dental sac at the bell stage(E16.5 d,E18.5 d and P0.5 d).4.(1)P0.5d EMSCs were isolated and cultured in vitro,and they were all smiliar to fiber cell and they have plump cell body and stronger cell proliferation ability.flow cytometry detection: The expression rate of CD14,CD29,CD90,CD146,CD166,p75 NTR of P0.5d EMSCs were respectively 92.93%,98.39%,95.49%,92.86%,91.44%,22.53%.The expression of CD45 of P0.5d EMSCs was respectively 5.78%.P0.5d EMSCs all positively expressed of CD14,CD29,CD90,CD146,CD166,p75 NTR,and expression and CD45 was negative.(2)After mineralized induced for 7d,14 d,21d d(0d as control),the results of p0.5d western blot method showed that expressions of p75 NTR and RUNX2 both increased with the prologation of mineralization induction time,which may has a positive correlation with each other(r=0.992,P<0.001).ConclusionsThe changes of locations in different days and expression quantities in p75 NTR and RUNX2 at the early stage of tooth had time-space specificity,which may be relate to mineralization of tooth.Tendency of changes verging to consistency after mineralization induction showed that p75 NTR and RUNX2 play very import role in development and mineralization of SD rats mandibular first molars at early developing stage,which may has a positive correlation with each other.