| Tooth eruption is a complex and tightly regulated process involved in both odontogenic and osteogenic cells. The dental follicle, a loose connective tissue sac surrounding each unerupted tooth, has been demonstrated to play an important role in this process. A major reason for its requirement is that it provides the eruption molecules, such as colony-stimulating factor-1 (CSF-1) and monocyte chemotactic protein-1 (MCP-1) etc. These molecules can promote the influx of mononuclear cells (monocytes) into the dental follicle to form osteoclasts which are needed to resorb alveolar bone for the eruption pathway.Recently, Wise et al found that in the rat first mandibular molar, a maximal burst of osteoclastogenesis was seen at day 3 postnatally, the time of peak expression of CSF-1 and MCP-1 in the rat dental follicle. It suggested that the major burst of osteoclastogenesis was closely related to CSF-1 and MCP-1. However, Wise et al further found that there was a minor secondary increase of osteoclasts at day 10 postnatally in the rat first mandibular molar. While at day 10 postnatally, CSF-1 and MCP-1 were weakly expressed. Thus it suggested that other molecules may be expressed by the dental follicle at this later time that would promote osteoclast formation. As the maximal expression of vascular endothelial growth factor (VEGF) in the rat dental follicle was at day 10 postnatally, it was postulated that VEGF may be a candidate molecule responsible for the minor burst of osteoclastogenesis at day 10. Their succedent work confirmed this postulation.However, these findings by Wise et al were based on rodentine experiments. It is not clear whether the conclusions obtained from rodentine dental follicle are applicable to the human dental follicle. Moreover, previous studies about the role of VEGF in tooth eruption lacked of systematicness. Many questions are still unknown, for example: the expression of VEGF in human dental follicle cells (HDFC), the biological effect of VEGF on HDFC, the effect of other eruption molecules on the expression of VEGF in cultured HDFC, the possible signaling pathway involved in regulating the expression of VEGF in cultured HDFC, the effect of VEGF on the expression of OPG and RANKL in cultured HDFC etc.Based on the previous findings, the present study is designed to systematically investigate the role of VEGF in tooth eruption with the use of the methods in cell biology and molecular biology. The present study will investigate whether VEGF is expressed in cultured HDFC. If VEGF is expressed in cultured HDFC, which signaling pathways are involved in regulating the expression of VEGF in cultured HDFC? Then the roles of VEGF in the proliferation, differentiation, and apoptosis of HDFC and its possible mechanism will be explored. The present study will also study the possible mechanism of VEGF involved in osteoclastogenesis. The results of the present study will help to elucidate the molecular regulation mechanism of tooth eruption and provide theoretical foundation for the orthodontic therapy of impacted teeth and tissue engineering research of tooth organs.The contents of the present study are as follows:Part 1. Expression of VEGF in cultured HDFCObjective: To investigate the expression of VEGF in cultured HDFC. Methods: The human dental follicle was separated from an impacted mandibular third molar extracted for orthodontic reasons from a 12-year-old boy. The primary HDFC were cultured by tissue culture plus trypsinization. The cells were observed and the expression of vimentin and cytokeratin were detected in the 4th passage HDFC by immunocytochemistry. Immunocytochemistry, enzyme linked immunosorbant assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression and transcription of VEGF in cultured HDFC. Results: HDFC were successfully cultured in vitro. The cultured HDFC displayed typical fibroblast-like morphological features. In the cultured HDFC, anti-vimentin immunostaining was positive while anti-cytokeratin immunostaining was negative. It suggested that the cultured HDFC derived from ectomesenchyme. Immunostaining for VEGF in cultured HDFC showed that brown immunostaining was localized in the cytoplasm, particularly in the perinuclear region. VEGF mRNA was successfully detected by RT-PCR and VEGF protein secretion was also detected by ELISA. Conclusions: HDFC can be cultured in vitro and these cells derive from ectomesenchyme. VEGF is transcribed and expressed in cultured HDFC.Part 2. The biological role of VEGF on the cultured HDFCObjective: To examine the roles of VEGF in the proliferation, differentiation, and apoptosis of HDFC in vitro. Methods: The 4th passage HDFC were seeded into 96-well plates. The dose-dependent and the time-course effect of VEGF on cell proliferation and alkaline phosphatase (ALP) activity in cultured HDFC were determined by MTT assay and colorimetric ALP assay, respectively. The effect of specific mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0126) on the VEGF-mediated HDFC proliferation was also determined by MTT assay. The effect of VEGF on HDFC apoptosis was measured by flow cytometry. Results: VEGF at 10~300ng/mL significantly increased HDFC proliferation and ALP activity compared to the control. Following 1, 3, 5, or 7d of stimulation, VEGF induced a significant increase in HDFC proliferation compared with the corresponding control, while VEGF was effective at increasing ALP activity at the incubation time point of 3, 5, or 7d. PD98059 and U0126 could attenuate the VEGF-mediated HDFC proliferation. Fewer apoptotic cells were observed in the VEGF-treated groups compared to the controls, although the difference was not statistically significant. Conclusions: VEGF at a proper concentration range can stimulate HDFC proliferation, induce HDFC to differentiate in a"cementoblast/osteoblast"pathway and protect HDFC from apoptosis. The MAPK signaling pathway might be involved in the VEGF-mediated HDFC proliferation.Part 3. Effect of TNF-αand bFGF on the expression of VEGF in cultured HDFCObjective: To study the effect of TNF-αand bFGF on the expression of VEGF in cultured HDFC. Methods: Concentration–dependent and time-course effect of TNF-αand bFGF on VEGF mRNA and VEGF protein secretion were determined by RT-PCR and ELISA respectively. Results:①In the concentration-dependent study, VEGF mRNA expression was increased after 1h exposure to various concentrations of TNF-αcompared with the control group (P<0.05), the optimal concentration was 25ng/mL. VEGF protein secretion was significantly increased after 12h exposure to various concentrations of TNF-αexcept TNF-αat 1ng/mL compared with the control group (P<0.05).②In the time-course study, using 25ng/mL TNF-α, 1h of incubation caused maximal expression of VEGF mRNA. The amount of VEGF mRNA decreased gradually after 3 or more hours of incubation as compared to 1-h treatment, but it was still greater than the control level (P<0.05). VEGF protein secretion was time-dependently increased, but VEGF protein levels in TNF-αtreatment groups were significantly higher than the control group (P<0.05).③In the concentration-dependent study, VEGF mRNA expression was increased after 6h exposure to various concentrations of bFGF compared with the control group (P<0.05), the optimal concentration was 10ng/mL. VEGF protein secretion was significantly increased after 12h exposure to various concentrations of bFGF (P<0.05).④The amount of VEGF mRNA increased significantly after 1 or more hours of incubation as compared with 0-h treatment (P<0.01). 6h of incubation caused maximal expression of VEGF mRNA. VEGF protein secretion was time-dependently increased, but VEGF protein levels in bFGF treatment groups were significantly higher than the corresponding control (P<0.01). Conclusions: Both TNF-αand bFGF can up-regulate the expression of VEGF in cultured HDFC.Part 4. Regulation of gene expression of VEGF in cultured HDFC via protein kinase C (PKC) and protein kinase A (PKA) signaling pathwaysObjective: To study whether PKC and PKA signaling pathways are involved in regulation of gene expression of VEGF in cultured HDFC. Methods: HDFC in good status were incubated with PMA (PKC activator), PMA+G?6983 (PKC non-specific inhibitor), PMA+ HBDDE (PKC-αandγspecific inhibitor), PMA+LY333531 (PKC-βspecific inhibitor), dbcAMP (PKA activator), dbcAMP+ KT5720 (PKA inhibitor) for 2h respectively. Real-time PCR was used to detect the gene expression of VEGF in these groups. Results: The gene expression of VEGF in the group with PMA alone or the group with PMA+ HBDDE was significantly higher than that of the control (P<0.05). There was no significant difference in the gene expression of VEGF between the group with PMA+G?6983 (or the group with PMA+LY333531) and the control (P>0.05). dbcAMP can up-regulate the gene expression of VEGF in cultured HDFC, while this effect can be inhibited by KT5720. Conclusions: The gene expression of VEGF in cultured HDFC can be regulated via PKC and PKA signaling pathways. In PKC signaling pathway, PKC-βmay be the isoform that regulates VEGF expression in cultured HDFC.Part 5. Effect of VEGF on the expression of OPG and RANKL in cultured HDFCObjective: To study the effect of VEGF on the expression of OPG and RANKL in cultured HDFC. Methods: HDFC in good status were incubated with 25ng/mL VEGF for different periods of time. The effect of VEGF on OPG mRNA and RANKL mRNA were determined by RT-PCR. The effect of VEGF on OPG protein secretion was detected by ELISA. Results: The amount of OPG mRNA decreased while the amount of RANKL mRNA increased after 1 or more hours of incubation as compared with the control. 3h of incubation caused minimal expression of OPG mRNA and 6h of incubation caused maximal expression of RANKL mRNA. OPG protein secretion was time-dependently increased, but OPG protein levels in VEGF treatment groups were significantly lower than the corresponding control (P<0.05). Conclusions: VEGF can down-regulate the expression of OPG and up-regulate the expression of RANKL in cultured HDFC. |
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