The Effect Of N-cadherin On The Proliferation, Migration And Odontoblastic Differentiation Of Human Dental Pulp Stem Cells In Vitro

Background and objectivePulp-dentine complex functions as a whole, and it possesses the ability to repair itself and regeneration. When suffering from the external irritants caused by infection, trauma or other physical and chemical factors, dental pulp stem cells residing in the pulp can proliferate and migrate to the lesion sites where they differentiate into odontoblast-like cells, and then secret dentine matrix and eventually form reparative dentine to protect dental pulp from advanced damages. However, it may develop into irreversible pulpitis or periapical diseases when the stimulants sustains and exceeds the threshold at which the pulp can endure. Currently, the main therapy for this situation is root canal treatment. It may help to control the infection and promote the healing of periapical diseases. Given the fact that root canal treatment can not restore the structure and function of the pulp-dentine complex, and thanks to the great progress achieved in the research of dental pulp biology, the concept of regenerative endodontics is emerging recently. Actually, pulp revascularization have been used in treating the immature permanent teeth clinically. However, reports showed that the tissue formed in the canals appeared to be of periodontal origin rather than pulpal origin and no pulp-like tissue characterized by the presence of odontoblast-like cells was observed lining the dentin-like mineralized tissue. Therefore, this technique would be a clinical success but a biological failure.Dental pulp stem cells (DPSCs) are mesenchymal stem cells separated from dental pulp tissues which possesses the capacity to self-renewal and multilineage differentiation. The odontoblastic differentiation of DPSCs are of great importance both in the process of dental pulp repair and regeneration. And the involving mechanism are so complicated that it remains elusive to date.Cell-cell and cell-matrix interactions mediated by cell adhesion molecules are important mechanisms controlling cell fate and function. Cadherins are transmembrane glycoproteins that mediate calcium dependent cell-cell adhesion.The cadherin superfamily can be divided into classical cadherins, desmosomal cadherins, protocadherins, seven pass transmembrane cadherins, and FAT-like cadherins. N-cadherin is a classical member of the cadherin super-family. Besides mediating cell-cell adhesion, N-cadherin can interact with lots of molecules and participate in many biological processes such as cell proliferation, apoptosis, migration and differentiation. The current research about N-cadherin mainly focus on mediating the tumor cell metastasis and the osteoblast differentiation. It has been reported that N-cadherin interacts with the fibroblast growth factor receptor (FGFR) and attenuates its ligand-induced downregulation, resulting in sustained mitogen-activated protein kinase/extracellular regulated protein kinases (MAPK/ERK) activation, matrix metalloprotein-9 (MMP-9) gene transcription, and cellular invasiveness. And N-cadherin can interact directly with the Wnt co-receptors low density lipoprotein receptor-related protein 5/6 (LRP5/LRP6) in osteoblasts, resulting in inhibition of Wnt/p-catenin signaling, osteoblast differentiation, as well as bone formation.N-cadherin was reported to be involved in the process of tooth development and dental pulp repair. During the human teeth development, N-cadherin was expressed in the differentiating and functional odontoblasts. Moreover, N-cadherin was re-expressed in odontoblasts surrounding the carious and traumatic sites. These results suggested that N-cadherin may play a critical role in tooth development and dental pulp repair. However, controversy did exist in whether N-cadherin is expressed in the dental pulp cells. And little information is available about the effect of N-cadherin on the biological function of human dental pulp stem cells (hDPSCs). Therefore, this study aims to confirm the expression of N-cadherin in hDPSCs, and investigate the role of N-cadherin in regulating the proliferation, migration and odontoblastic differentiation of hDPSCs.Methods1. The expression of N-cadherin in human dental pulp stem cellsHealthy human premolars or third molars extracted due to orthodontic or impaction from individuals (13-25 years old) were collected and hDPSCs were enzymatically separated from dental pulp tissues, cultured, and passaged. To confirm the tissue origin of hDPSCs, the expression of vimentin and cytokeratin was detected by cytoimmunochemistry. Flow cytometry was utilized to analyze the expression of hDPSCs surface proteins. To research the multilineage differentiation capacity, hDPSCs were conducted to odontoblastic medium containing 10 nM dexamethasone, 50 mg/L ascorbic acid and 10 mM P-glycerophosphate for 14 days, and Alizarin red staining was used to examined the mineralization nodules formation. Meanwhile, hDPSCs were conducted to chondrogenic medium consisting of 1 μM dexamethasone, 200 μM idomethacin,0.5 mM 3-isobatyl-l-methylxanthine (IBMX) and 10μg/mL insulin for 21 days, and Oil red O staining was utilized to visualize the lipid vacuoles formation.hDPSCs were lysed to extract total RNA by using TRIzol and the expression of N-cadherin mRNA in hDPSCs was determined by reverse transcription polymerase chain reaction (RT-PCR). Cytoimmunochemistry was used to reveal the location and expression of N-cadherin protein in hDPSCs. On the other hand, hDPSCs grown in odontoblastic medium or control medium were lysed at day 7 and 14 respectively, Real time quantitative polymerase chain reaction (Real time PCR) was performed to detect the expression profile of N-cadherin in hDPSCs after the odontoblastic induction.2. The effect of N-cadherin knockdown on the odontoblastic differentiation of human dental pulp stem cellsSpecific N-cadherin shRNA lentivirus was constructed, and the optimal conditions for lentivirus transfection was investigated. A total of 12 groups were established according to the different combination of complete medium (CM) or enhanced infection solution (Enis), MOI (10,25,50), polybrene (5 μg/mL). Transfection efficiency of WT, ShRNA-Ctrl and ShRNA-N-cad group hDPSCs were observed under fluorescence field by inverted phase contrast microscopy after lentivirus transfection for 72 hours. The knockdown of N-cadherin both in mRNA and protein level after lentivirus transfection were confirmed by Real time PCR and Western blot, respectively.WT, ShRNA-Ctrl and ShRNA-N-cad group hDPSCs were conducted to odontoblastic medium, the expression of odontoblastic-related genes alkaline phosphatase (ALP), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and potential signal molecule β-catenin were detected by Real time PCR and Western blot. Moreover, the ALP activity and mineralization nodules formation capacity were detected by ALP activity staining at day 7 and Alizarin red staining at day 14, respectively.3. The effect of N-cadherin knockdown on the proliferation and migration of human dental pulp stem cellsTo elucidate the effect of N-cadherin knockdown on the proliferation of hDPSCs, WT, ShRNA-Ctrl and ShRNA-N-cad group hDPSCs were seeded in 96-well plates, the optical density (OD) were measured at 450 nm from day 1 to 8 by CCK-8 assay. Furthermore, flow cytometry was utilized to analyze the cell cycle and cell apoptosis.And to investigate the role of N-cadherin suppression in the migration of hDPSCs, Transwell assays was conducted. Briefly, hDPSCs were harvested and counted, and 5×104 cells of each group were seeded to the upper chamber with 200 μL basic medium without FBS, whereas 600 μL complete medium containing 100 mL/L FBS were added to the lower chamber. After incubation for 20 hours, the cells migrated through the membrane pores were counted. Besides, the expression of related chemokines stromal cell-derived factor-1 (SDF-1) and CXC receptor 4 (CXCR4) were detected using Real time PCR.Results1. The culture and identification of human dental pulp stem cellsThe hDPSCs cultured in vitro grew well and demonstrated fibroblast-like morphology. Cytoimmunochemistry staining showed that the gained hDPSCs were positive to vimentin and negative to cytokeratin. The flow cytometry analysis showed that the hDPSCs expressed the mesenchymal cell surface proteins CD44, CD90, CD 29 (100%,99.9%,94.7%, respectively), and did not express the hematopoietic cell surface proteins CD45, CD31, CD34 (0.47%,0.13%,0.16%, respectively). These results indicated that the hDPSCs in this study were mesenchymal origin and not from epithelial or hematopoietic cells. After odontoblastic and chondrogenic induction, hDPSCs could form mineralization nodules and lipid vacuoles as detected by Alizarin red staining and Oil red O staining, respectively, suggesting that hDPSCs possessed the multilineage differentiation capacity.2. The expression of N-cadherin in human dental pulp stem cellsRT-PCR showed that N-cadherin mRNA was expressed in hDPSCs cultured in vitro. Moreover, cytoimmunochemistry staining revealed that N-cadherin was mainly expressed in the cytomembrane and cytoplasm of hDPSCs. Interestingly, the expression of N-cadherin was significantly downregulated in hDPSCs after the odontoblastic induction.3. Knockdown the expression of N-cadherin in human dental pulp stem cellsThe specific N-cadherin shRNA lentivirus was successfully constructed and the optimal conditions for lentivirus transfection was complete medium containing 5 μg/mL polybrene with MOI 25. The transfection efficiency could reach up to 80% as visualized under fluorescence field by inverted phase contrast microscopy. Furthermore, Real time PCR and Western blot results showed that N-cadherin were remarkly knockdown both in mRNA and protein level after N-cadherin shRNA lentivirus transfection.4. The effect of N-cadherin knockdown on the odontoblastic differentiation of human dental pulp stem cellsAfter odontoblastic induction, Real time PCR and Western blot indicated that, compared with ShRNA-Ctrl group, the expression of odontoblastic-related genes ALP, OCN, DMP1, DSPP as well as β-catenin were significantly upregulated in ShRNA-N-cad group. Meanwhile, the ALP activity and mineralization nodules formation were increased in ShRNA-N-cad group.5. The effect of N-cadherin knockdown on the proliferation of human dental pulp stem cellsCCK-8 assay revealed that, compared with ShRNA-Ctrl group, the proliferation activity increased at the early stage and decreased at the late stage with a turning point at day 5 in ShRNA-N-cad group. To further confirm whether the ShRNA-N-cad group hDPSCs exhibited decreased proliferation or were undergoing apoptosis at the late stage, we conducted cell cycle and apoptosis assays at day 6. The results suggested that the knockdown of N-cadherin blocked the cell cycle at the G0/G1 phase and reduced rate of entrance into the S and G2/M phases. Cellular apoptosis analysis revealed that more cells were undergoing apoptosis because of N-cadherin shRNA lentivirus treatment.6. The effect of N-cadherin knockdown on the migration of human dental pulp stem cellsTranswell assays demonstrated that the cells migrated through the membrane pores in ShRNA-N-cad group were more than that in ShRNA-Ctrl group. Moreover, the expression of related chemokines SDF-1/CXCR4 were significantly upregulated in ShRNA-N-cad group compared with ShRNA-Ctrl group as detected by Real time PCR.Conclusions1. The hDPSCs were successfully cultured in vitro, and they were mesenchymal origin, as well, possessed the potential to undergo odontoblastic and chondrogenic differentiation.2. N-cadherin was expressed in hDPSCs cultured in vitro, and it was mainly expressed in the cytomembrane and cytoplasm of hDPSCs.3. The expression of N-cadherin in hDPSCs was significantly downregulared after N-cadherin shRNA lentivirus transfection.4. Knockdown of N-cadherin promoted odontoblastic differentiation of hDPSCs probably via activating β-catenin pathway.5. The effect of N-cadherin knockdown on the proliferation of hDPSCs was time-dependent.6. The promotion effect of N-cadherin knockdown on the migration of hDPSCs was potentially mediated by SDF-1/CXCR4 upregulation.