The Mechanism Underlying The Osteogenic Induction Of Dental Follicle Stem Cells By CA1 In Vitro

BACKGROUND AND OBJECT Cleft lip with or without cleft palate(CL/P)is one of the most common congenital malformations,accompanied by negative effects on language,hearing,appearance and psychology,which can lead to long-term adverse consequences of patients’ social integration.Patients with CL/P need long-term sequential treatment,in which bone grafting of alveolar cleft is one of the most crucial and difficult points in sequential treatment of CL/P.In this study,lentiviral transfection,RNA interference and other research techniques were used to regulate the expression of Carbonic anhydrase1(CA1)during the osteogenic process of Dental follicle stem cells(DFSCs)in vitro.The purpose of this study is to investigate the role and mechanism of CA1 in the osteogenic induction of DFSCs in vitro,and to lay a theoretical and experimental foundation for in vivo experiments and clinical application of bone tissue engineering for alveolar bone grafting.METHODS1.DFSCs from human dental follicle tissues were isolated by enzymatic digestion and its expression surface markers of mesenchymal derived cells demonstrated by flow cytometry.Alizarin red staining and oil red O staining were used to identify the differentiation potential of osteogenic and adipogenic cells.2.Small interfering RNA si-CA1 was used to transfect DFSCs during osteogenic induction culture.Western blot(WB)and Quantitative Real-time PCR(q RT-PCR)were used to detect the m RNA and protein expression of CA1,alkaline phosphatase(ALP),Bone morphogenetic protein 2(BMP 2)and Runt-related transcription factor 2(RUNX2)in si-CA1 group,negative control group and blank control group.3.Lentiviral vector LV-CA1,which overexpressed CA1 gene,was constructed to transfect DFSCs during osteogenic induction culture in vitro.q RT-PCR and WB were used to detect the m RNA and protein expressions of CA1,ALP,RUNX2 and BMP2 in LV-CA1 group,negative control group and blank control group after transfection.Alizarin red staining was used to evaluate the osteogenic effect.RESULTS1.DFSCs were successfully obtained from dental follicle tissue by enzymatic digestion,and the cells showed significant osteogenic and adipogenic differentiation potential.Flow cytometry showed high expression of CD29,CD73 and CD90,but no expression of CD45,which was consistent with the phenotype of mesenchymal cells.2.Compared with negative control group and blank control group,m RNA and protein expression of CA1 in si-CA1 group were significantly decreased(P < 0.05).The m RNA and protein expressions of osteogenic markers ALP,RUNX2 and BMP2 were also decreased to varying degrees(P<0.05).3.Compared with negative control group and blank control group,m RNA and protein expression of CA1 in LV-CA1 group were significantly increased(P < 0.05).The m RNA and protein expressions of osteogenic marker genes ALP,RUNX2 and BMP2 were increased in different degrees(P<0.05).In addition to CA1,BMP2(43.01%)and ALP(36.69%)were significantly increased(P<0.05).In the 28-day osteogenic induction culture,the expression level of CA1 in the 3 groups of cells showed a trend.At the initial stage of culture(day 3),it went slightly higher,then decreased at day 7,and gradually increased at the later stage.The highest expression level was found at day 21.Alizarin red staining results indicated that LV-CA1 group had more calcified nodules,higher mineralization degree and better osteogenic effect compared with negative control and blank control group at the same time point(P<0.05).CONCLUSIONIn this study,human DFSCs were cultured in vitro.In the osteogenic induction culture process using DFSCs,the expressions of ALP,RUNX2 and BMP2 can be regulated by increasing or decreasing CA1 expression,and the former can achieve better osteogenic effect than control groups.During osteogenic induction culture,the expression level of CA1 increased on day 3 and 21 of induction,especially at the later stage of osteogenesis with calcified nodules forming.These results revealed that CA1 could affect the osteogenic differentiation process through osteogenic marker genes(ALP,RUNX2,BMP2)in BMP signaling pathway,and also promote the in vitro osteogenesis of DFSCs.This study provides an experimental basis for further application of CA1 and DFSCs in bone tissue engineering to repair alveolar cleft.