iTRAQ-Based Proteomic Analysis Exploring the Influence of Hypoxia on the Proteome of Dental Pulp Stem Cells under 3D Culture.

Hypoxic preconditioning is commonly applied to enhance mesenchymal stem cells (MSCs) therapeutic effect before transplantation. Elucidating the effect of hypoxic preconditioning would be beneficial for improved application. However, the influence of hypoxia on dental tissue derived MSCs cultured in 3D was unknown. Thus, the present study is to investigate gene expression and proteome of dental pulp stem cells (DPSCs) after hypoxic preconditioning. DPSCs were isolated, cultured in a 3D system under the normoxic and hypoxic conditions. The gene expression was examined with reverse transcription polymerase chain reaction, and the proteome was analyzed using iTRAQ-based mass spectrometry. The expressions of HIF-1α, VEGFA, KDR at mRNA level was upregulated while BMP-2 was downregulated. Two thousand one hundred and fifteen proteins were identified and 57 proteins exhibited significant differences after hypoxic preconditioning (30 up-regulated, 27 down-regulated). Bioinformatic analysis revealed the majority of up-regulated proteins are involved in cellular process, angiogenesis, protein binding and transport, regulation of response to stimulus, metabolic processes, and immune response. Increased IL-6 and decreased TGF-1ß protein expression under hypoxic condition were verified by ELISA. Hypoxic preconditioning partly affected the gene and protein expression in DPSCs under 3D culture and may enhance the efficacy of MSCs transplantation.

[The Effect of Concentrate Growth Factors on the Survival, Proliferation and Differentiation of Human Dental Pulp Cells].

OBJECTIVE: To explore the cytotoxicity of concentrate growth factors (CGF) and the effects on the apoptosis, proliferation and differentiation of human dental pulp cells (hDPCs), which were closely correlated with future application of CGF in the treatment of dental pulpal and periapical diseases. METHODS: hDPCs were isolated from permanent teeth extracted for orthodontic purpose, and expanded in vitro. hDPCs were treated with CGF and mineral trioxide aggregate (MTA) respectively. The cell apoptosis, proliferation, cell cycle and ALP activity were analyzed after 1, 3 and 7 days. RESULTS: Compared with the MTA group, CGF significantly promoted cell proliferation, increased the proportion of S-phase cells and ALP activity on days 3 and 7 (P<0.01). Besides, hDPCs apoptotic rates decreased in CGF group. CONCLUSION: CGF has a good ability to promote the proliferation of dental pulp cells, resist apoptosis and induce osteogenic/odontogenic differentiation in vitro.

Effect of five dental pulp capping agents on cell proliferation, viability, apoptosis and mineralization of human dental pulp cells.

The aim of the present study was to investigate the effect of calcium hydroxide [Ca(OH)2], mineral trioxide aggregate (MTA), iRoot BP, platelet-rich fibrin (PRF) and concentrated growth factors (CGF) on the proliferation, viability, apoptosis and mineralization of human dental pulp cells (HDPCs). HDPCs were treated with Ca(OH)2, MTA, iRoot BP, PRF and CGF exudates. Cell viability, apoptosis, proliferation, cell cycle and alkaline phosphatase (ALP) activity were evaluated in vitro. PRF significantly increased the cell proliferation as compared with that in the MTA and iRoot BP groups on day 3. The CGF group displayed higher proliferation rates as compared with that in the MTA group on days 3 and 7. The MTA group displayed the highest ALP activity on days 1 and 3, and the CGF group on day 7. Ca(OH)2 inhibited cell proliferation and the percentages of dead and apoptotic cells were relatively higher in the Ca(OH)2 group on days 1, 3 and 7 compared with those in the other groups. In conclusion, PRF and CGF may be potential pulp-capping materials for vital pulp therapy. Future in vivo studies are required to confirm this.

[An in vitro study of the angiogenic effects of concentrate growth factor on human umbilical vein endothelial cells].

OBJECTIVE: This study aimed to explore the effects of concentrate growth factor extracts (CGFe) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Concentrate growth factor (CGF) were prepared from the peripheral blood of healthy donors, followed by CGFe. Four groups were designed based on cell culture medium, as follows: 2%CGFe, 5%CGFe, 10%CGFe, and control. The proliferation activity of HUVECs was detected by cell cycle and CCK-8 assays. The migration of HUVECs was detected by scratch assay. The mRNA expression levels of vascular endothelial growth factor (VEGF), chemokine receptor 4 (CXCR4), and platelet derived growth factor (PDGF) were examined by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: Results of CCK-8 and cell cycle assays showed that CGFe promoted the proliferation capability of HUVECs in a dose-dependent manner, and the data had statistical significance among four groups (P<
0.05). The cell migration assay indicated that CGF accelerated wound closure in a dose-dependent manner after 12 h of culture (P<0.05). The results of qRT-PCR showed that CGF upregulated the expression levels of VEGF, CXCR4, and PDGF in HUVECs. CONCLUSIONS: CGFe can promote the proliferation, migration, and angiogenic differentiation of HUVECs. Thus, CGF might be an appropriate cure for dental pulp revascularization.

Secretome profiles of immortalized dental follicle cells using iTRAQ-based proteomic analysis.

Secretomes produced by mesenchymal stromal cells (MSCs) were considered to be therapeutic potential. However, harvesting enough primary MSCs from tissue was time-consuming and costly, which impeded the application of MSCs secretomes. This study was to immortalize MSCs and compare the secretomes profile of immortalized and original MSCs. Human dental follicle cells (DFCs) were isolated and immortalized using pMPH86. The secretome profile of immortalized DFCs (iDFCs) was investigated and compared using iTRAQ labeling combined with mass spectrometry (MS) quantitative proteomics. The MS data was analyzed using ProteinPilotTM software, and then bioinformatic analysis of identified proteins was done. A total of 2092 secreted proteins were detected in conditioned media of iDFCs. Compared with primary DFCs, 253 differently expressed proteins were found in iDFCs secretome (142 up-regulated and 111 down-regulated). Intensive bioinformatic analysis revealed that the majority of secreted proteins were involved in cellular process, metabolic process, biological regulation, cellular component organization or biogenesis, immune system process, developmental process, response to stimulus and signaling. Proteomic profile of cell secretome wasn't largely affected after immortalization converted by this piggyBac immortalization system. The secretome of iDFCs may be a good candidate of primary DFCs for regenerative medicine.