Effects Of Wnt5a On Odonto/Osteogenic Differentiation Potential Of Human Stem Cells From The Apical Papilla In Vivo

Objective: The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a were established, the purpose of this study was to discuss the effects of Wnt5 a on Odonto/ Osteogenic differentiation potential of Human Stem Cells from the Apical Papilla in vivo, which would facilitate a certain theoretical and experimental support for the application of Wnt5 a effects on Human SCAP in pulp/dentin regeneration and bioroot formation.Methods: Part 1: Construction and identification of the stable tansfection SCAP cell line of overexpression and silent of Wnt5 a Human stem cells from the apical papilla were primary cultured and isolated by enzyme-digestion method and limited dilution technique, in vitro. The isolated cells were investigated for the expression of mesenchymal stem cell markers and evaluating their multidifferentiation potentials by immunoctofluorescent stainning technique and osteogen-ic, adipogenic, induction in vitro. The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a were constructed respectively by using Wnt5 a overexpression and silent lentiviral vector. q RT-PCR and Westernblot were used to detect the expression of Wnt5 a m RNA and protein in the cell line of Wnt5a-SCAP and SCAP respectively. Part 2: Effects of Wnt5 a on Odonto/Osteogenic differentiation potential of SCAP in vivo The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a, and SCAP were cultured respectively in three-dimension scaffold of HA/TCP, and then the biologicalcharacteristics of the cells were observed through the scanning electron-microscope. The co-cultured mixture of SCAP and hydroxyapatite/tricalcium phosphate(HA/TCP) was implanted into the subcutaneous space of immunocompromised mice for 4 weeks and 8 weeks respectively. Then the implants were collected and odontoblast 1ike cells, odontoblat like substrate and dentin and pulp composition were examined by HE and Masson’s Trichrome staining. The osteo/odontogenic markers including alkaline phosphat-ase(ALP), bone sialoprotein(BSP), dentin sialophosphoprotein(DSPP) and osteocalcin(OCN) in the SCAP implantation tissue were investigated by immunohistochemistry staining.Results: 1 The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a were constructed successfully. SCAP were primary cultured, isolated and purified successfully by enzyme-diges-tion method and limited dilution technique, in vitro. SCAP possessed the characteristics of mesenchymal stem cell, which were investigated by immunofluorescent stainning techni-que; It has multidifferentiation potentials by the induction differentiation technique of osteomenic and adipogenic as well. The SCAP were infected by the retroviruses lentiviral of overexpression and silent Wnt5 a lentiviral vector at the drop degree of the 107(MOI=10). After being selected and purified, the stable tansfection SCAP cells colony of Wnt5 a were gained successfully infected with Wnt5 a screening lentiviral, the transfection rate of which was above 90%. The Expression levels of Wnt5 a m RNA and protein were confirmed by q RT-PCR and Western blotting; Compared with its relevant empty vector control respectively, the expression levels of Wnt5 a m RNA and protein increasesed in Wnt5 a overexpression groups, while it was downregulated in slient Wnt5 a goups(P<0.05). 2 The effects of Wnt5 a on Odonto/Osteogenic differentiation potential of SCAP in vivo(1) The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a and its relevant empty vector control and SCAP were cultured for 24 hours respectively in three-dimension scaffold of HA /TCP, and then the biological characteristics of the cells were observed through the SEM. It showed that the cell line of groups could attach on the surface and the hole of HA/TCP, extending well and forming cell sheet struture respectively.(2) The implants were collected and examined by HE and Masson’s Trichrome staining. Result showed that odontoblast 1ike cells differentiation, matrix mineralization, odontoblast like substrate, dentin and pulp composition like substrate could be seen obviously in Wnt5 a overexpression groups, whereas there was only the odontoblast /osteogenic 1ike cells differentiation, little matrix mineralization and odontoblat like substrate could be seen in Wnt5 a slient goups.(3) The immunohistochemistry staining showed that the osteo/odontogenic markers including alkaline phosphatase(ALP), dentin sialo phosphoprotein(DSPP), bone sialoprotein(BSP), and osteocalcin(OCN) in the SCAP implantation tissue were expressed at differrent extent in control groups, overexpression Wnt5 a groups and slient Wnt5 a goups. It was strongest in all group at the fourth week, which was stronger in Wnt5 a overexpression groups than that of in empty vector and blank control groups; and less in the slient goups than empty vector and blank control groups, while the expression of the osteo/odontogenic markers in overexpressed Wnt5 a group was higher than slient Wnt5 a group.At the eighth weeks, the ALP, DSPP and BSP expression in Wnt5 a silent groups was higher than that of Wnt5 a overexpression groups and its empty vector control groups, but there was no significant difference in OCN between the groups.Conclusion: 1 The stable tansfection SCAP cell line of overexpression and silent of Wnt5 a were constructed successfully, the transfection rate of which was above 90%. 2 The SCAP cell line of overexpression and silent of Wnt5 a could attach on the surface and the hole of HA/TCP, cextending well and forming cell sheet struture respectively, which showed that there was good biocompatibility between each other, The co-cultured mixture of HA/TCP carrier mixed with overexpression and silent of Wnt5 a SCAP and its relevant empty vector control screening lentiviral were constructed successfully. 3 Overexpression Wnt5 a could effectively induce SCAP to differentiat into odontoblast 1ike cells, promote matrix mineralization and the formation of the dentin-like tissue, dentin and pulp composition like substrate and so on, whereas the process of the odonto/osteogenic 1ike cells differentiation and thematrix mineralization in slient Wnt5 a goupswas inhibited. 4 Overexpressed Wnt5 a can promote the expression of odonto/osteogenic mineraliza-tion related genes ALP, BSP, DSPP, OCN in vivo, the expression of which were decreased in slient Wnt5 a group obviously at 4 weeks.5 Collectively, in this study, we have preliminary confirmed that Wnt5 a could promote odonto/osteogenic differentiation potential of SCAP in vivo. And Further confirmed that Wnt5 a palyed an important role in regulating odonto/osteogenic differentiation potential of SCAP, which provided a certain theoretical and experimental evidence for the application of Wnt5 a in the pulp/dentin regeneration, bioroot formation, and the molecular mechanisms of root development in the future.