The Biological Behavior Changes In Apical Papilla Cells From Human Supernumerary Teeth After Modification By Wnt5a Gene

ObjectiveApical papilla cells(APCs-S)and dental pulp cells(DPCs-S)from human supernumerary teeth were isolated and cultured in vitro to compare the difference of their biological behavior.Using lentivirus vector to make the wingless-type MMTV integration site family member 5a(Wnt5a)gene over-expression in APCs-S and investigate the biological behavior changes in APCs-S after modification by Wnt5 a gene.Methods1.Using enzymatic digestion method,APCs-S and DPCs-S were isolated and cultured in vitro.The optical density(OD)values were detected at 1st,3rd,5th and 7th day by CCK-8 assay.Alizarin red staining was applied to count the calcium nodules after osteoinduction of APCs-S and DPCs-S.Lipid droplets were observed by using oil red O staining of APCs-S and DPCs-S after adipogenic induction.2.Lentiviral vector was used to over express wingless-type MMTV integration site family member 5a(Wnt5a)gene in APCs-S.The antibiotic-resistant cells were selected by applying puromycin in culture medium.The messenger RNA(mRNA)expression of Wnt5 a genes in infected APCs-S was detected by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)technique.Infected APCs-S were cultured and osteogenic induction in vitro.BCIP/NBT staining was performed to detect the expression of alkaline phosphatase(ALP)in two groups.Alizarin red staining was applied to count the calcium nodules after osteoinduction.The mRNA expression of bone formation-related gene in two groups were detected by Real-time PCR technique.Results1.APCs-S were successfully isolated and cultured in vitro by using enzymatic digestion method.The cells of two groups had good morphology.The proliferation ability of APCs-S was better than DPCs-S.The osteogenic differentiation potential of APCs-S and DPCs-S were tested by using alizarin red staining.The adipogenic differentiation potential of APCs-S and DPCs-S were tested by using oil red O staining.2.Using lentiviral vector successfully made Wnt5 a gene over-expression in APCs-S.The Wnt5 a gene expression levels of Wnt5 a over-expression group were significantly higher than the negative control lentivirus group.BCIP/NBT staining showed that both groups could secrete ALP.Alizarin red staining showed that Wnt5 a gene over-expression in APCs-S promoted the formation of orange calcium nodules.After osteoinduction,the gene expression level of ALP,runt-related transcription factor 2(RUNX2)and bone sialoprotein(BSP)in APCs-S after modification by Wnt5 a gene was significantly increased.Conclusion1.Both APCs-S and DPCs-S showed active proliferative capacity and multi-lineage differentiation potential.Compared to DPCs-S,APCs-S showed higher proliferation and osteogenic differentiation capacity.APCs-S was more suitable to be the seed cells of gene engineering.2.Using lentiviral vector successfully made Wnt5 a over express in APCs-S.Wnt5 a gene can promote the osteogenic differentiation of APCs-S.