Effects Of Wnt5a And Igf1 On Odontogenic Potential Of Mouse Molar Mesenchyme

Exploring molecular mechanisms of the tooth germ tissue potential has great significance in tooth regeneration. In this study, we firstly tested the potential changes of E14.5 mice mandibular molar mesenchyme after 0h,24h and 36h of organ culture in vitro. The results showed that extending in vitro culture time decreased remarkably the tooth formation rate and delayed tooth development. The growth factors (Hgf, Igfl, Bmp3, Bmp5, Bmp6, Tgfbl, Bmp2, Bmp4, Bmp7, Wnt3, Wnt4, Wnt5a, Wnt6, WntlOa, WntlOb, Fgf9, Fgf10, Follistatin, ActivinβA, Midkine and Shh) effected in tooth development were analyzed by RT-PCR after E14.5 mice mandibular molar mesenchye was cultured in vitro for 24h, The results showed that t Bmp2 wasn’t expressed in molar mesenchye, the expression levels of Wnt3,Wnt10a,Wnt10b, Shh were slightly elecated, the expression levels of Hgf, Igf1, Bmp3, Bmp5, Bmp6, Activin βA, Midkine, Tgfb1, Bmp2, Bmp4, Bmp7, Wnt4, Wnt5a, Wnt6, Fgf9, Fgf10 and Follistatin were all decreased. That indicated that the loss of odontogenic potential of molar mesenchyme maybe are related to the decreased expression of growth factors after in vitro tissue culture.Weather overexpression of some growth factors in cultured molar mesenchyme can save partly its odontogenic potential? Three growth factors (Wnt5a, Activin βA and Igf1) with loss of their expression after culture were chosed for father overexpression research in 24h cultured E14.5 mandibular molar mesenchyme. Wnt5a and Igf1 gene can produce several transcript variants by alternative splicing except Activin βA gene. In order to determine transcript variants of Wnt5a and Igf1 gene transcripted in molar mesenchyme, the total RNA from E14.5 mandibular molar mesenchyme was extracted and analyzed by RT-PCR. The results showed that two transcript variants of Wnt5a (Wnt5a variant 1 and Wnt5a variant 2) and four transcript variants of Igf1 (Igf1 variant 1, Igf1 variant 3, Igf1 variant 4 and Igfl variants) were expressed in E14.5 molar mesenchyme. Activin βA, Wnt5a variant 1、Wnt5a variant 2 and Igfl variant 4 cDNAs were cloned into Tet-on inducible expression plasmid pTRE-DsRedl-IRES-EGFP AutoR3. The lentiviral particles expressing Wnt5a variant 1、Wnt5a variant 2 and Igfl variant 4 were successfully produced from 293 T cells, but lentiviral particles expressing Activin PA were fail to generate. E14.5 mouse mandibular molar mesenchyme after 24h of organ culture was transduced by Wnt5a variant 1、Wnt5a variant 2 and Igfl variant 4 expressing lentiviruses, respectively, or by Wnt5a variant 1 and Wnt5a variant 2 expressing lentiviruses simultaneously. After confirming the expression of these genes in molar mesenchymal cells, the mesenchymal cells transduced by lentiviruses was recombined with E14.5 mandibular molar epithelium, and then transplanted into the mice renal capsule in culture for 2 weeks. Compared to control group tranduced with empty lentiviral vector, tooth formation rate was increased slightly in overexpressing Wnt5a variant 1 or Wnt5a variant 2 groups, tooth formation rate wasn’t affected in overexpressing Wnt5a variant 1 and Wnt5a variant 2 simultaneously group. tooth formation rate was decreased slightly in overexpressing Igfl variant 4 group. Part of recombinant teeth were abnormality in structure with enamel loss in overexpressing Wnt5a variant 1 or/and Wnt5a variant 2 groups.