Optimization Of Culture System To Promote The Maintainance Of Odontogenic Potential Of Mouse Dental Mesenchymal Cells

Teeth are important organs in the process of human growth and development.Tooth injury or loss have become common diseases in stomatolog y.Tooth loss could lead to prob lems in ch ewing,langu age an d aesth etics,which would affect the quality of human life to varying degrees.Traditional dental restoration method s displ ay short service life,poor sense of use,poor biocompatibility and other problems.Therefore,how to obtain the regenerative teeth similar to natural teeth in function and morphology has become a hot topic in the field of stomatology.Tooth develop s from the interactio ns b etween the oral epith elium and the surrounding mesen chyme,which i s regulated by v arious signalin g pathway s.During odonto genesis,int eractions b etween tissues are manifested by the fact that one tissue capable of producing the induction signal and the other tissue capable of receiving and respondi ng to the signal.This induction ability of the former tissue is called odontogenic potential.It is well kno wn that the o dontogenic potential shifts from dent al epithelium to dental mesenchym e at bud stage(embryo day 12-13,E12-13).Thus,the early dental epithelium can induce dental or non-d ental mesen chyme to form teeth,while the dental mesench yme after bud stage can induce dent al or non-dental epithelium to form teeth.Based on thi s,mouse d ental mesen chymal cells at cap stage(E14.5)were widely appli ed as an induction sign al cell source to toot h regeneratio n.Howev er,the number of freshly i solat ed dent al mesench ymal cells i s limited,significantl y restricting the reg eneration st udies of teeth.Previousl y,we have identified that the mouse dental mesenchymal cell s(m DMCs)lose their odontogen ic potential after 2 4 hours during the in vit ro culture wit h MEF medium.The odontogenic pot ential co uld be retained till 48 hour s in the optimization syst em with KSR medi um(knockot serum replacement).In order to further maintain the odontogenic potential of m DMCs in vitro and to clarify the und erlying changes,we here optimized the culture syst em of m DMCs in vitro and analyzed the possibl e key factors for maintaining odontogenic pot ential by transcriptome sequencing,which may provid e clues for the study of tooth regenerati on.In this study,we set up a new culture system(NM)based on previous optimized serum-free condition.Com pared with MEF medium(DMEM+10%F BS)and KSR m edium(DME M+20%KS R),we observed t he growth of cells und er microscope an d detected th e expressi on of odontogenic-rel ated gen e expression s in m DMCs in three different culture condition s.The cultured cells were further reaggregated from different media at different time points and recomb ined with E14.5 mouse dental epithelium without odonto genic potential.The recombinants were then transplant ed into adult male mouse subr enal capsule.The grafts were dissected after 3 weeks and examin ed histolo gically.Moreover,transcriptome sequen cing was used to detect differences in the transcriptome of cultured DMCs with different culture media and analyze the mechani sm of odontogeni c potential mai ntenance.As results,cultured DMCs adhered to the plates and grew with the long spindle shap e in MEF and KSR mediu m conditions.Whereas,cultured DMCs were cluster ed in NM medium.Some cells were su spend ed in pellets after 4 days of culture,which indicated that NM serum-free medium promoted cell suspen sion culture and could simulate the three-dimen sional culture environment.The expression of genes related to odontogen esi s was detected by q PCR.It was found that the expressio n level s of Dlx2,Lhx6,Lhx8,Msx2 and Msx1 in NM group was higher than those in MEF and KSR groups.As the number of passages increased,the gene expression level s in NM decreased gradu ally.Especially,the expressio n of Msx1 was detected to decrease by 40%,indicating that the odontogenic ability of DMCs in NM graduall y lost during p assage.A ccording to the result s of cell recombination tran splantati on and histologi cal examination,cell s cultured for 1 day in MEF and KSR medium were respectively recombin ed with E14.5 dental epith elium and the graft s wer e detected to form teeth with a 100% success rate,whi le the cell s cultured for more than 1 day cann ot form teeth after recombination with E14.5 dental epith elium.On the other hand,the cells cultured for 1,4,and 7 days in NM could produce teeth after recombination with E14.5 dental epit h elium with success rate 83.3%,58.3% and 66.7% respectively.But these cells failed to form teeth after passage in NM either.Transcription sequen cing data displayed the obviou s differences betw een groups in different culture system s with different culture time point s.In particularly,a great difference in the expression of Bmp4 was detected between the odontogen ic group in 4-day culture with NM and the non-odo ntogeni c group in 4-day culture with MEF,showing a lower expression lev el of Bmp4 in the odontogenic group and a high er expression level of Bmp4 in the non-odonto genic group.In order to clarify the role of Bmp4 in odontogenic potential,we suppl emented BMP4 into th e NM odontogen ic group and BMP inhibitor DM(dorsomorphin)into MEF non-odo ntogeni c group resp ectively.We f uther harvest ed teeth from the MEF non-od ontogeni c group supplyment ed with DM at a su ccess rat e up to 50%,indicating the odontogenic potenti al o f cultured cells in MEF sy stem was resued partly.Neverth eless,no teeth were found in the NM odontogenic group added with BMP4,sug gesting BMP4 may inhibit th e maintainan ce of odontogeni c potential of D MCs in vitro.Taken together,our results d emonstr at ed that optimized N M serum-free medium can maintai n the odont ogenic potenti al of DMCs for 7 days in vitro through a 3 D microen vionmen t.This optimized sy stem co uld effectively improve th e rate of tooth formation after recombinati on with E14.5 dental epithelium.According to transcriptom e sequencing data,great differences of odontgenic relat ed gen e expression s were detected in the cutured cells before and after optimization.One of the differenti ally expressed genes,Bmp4,was selected for validation.And it was found that Bmp4 plays a key role i n the formation of tooth,and BMP4 inhibitor D M can restore th e odontog enic abilit y of cult ured cells.These result s may provide a new sight for the culture of DMCs in vitro and a possible way for regulation of activating signal s in the constr uction of regenerativ e teeth.