Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT.

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.

Differential effects of TGF-ß1 and FGF-2 on SDF-1α expression in human periodontal ligament cells derived from deciduous teeth in vitro.

Stromal cell-derived factor (SDF)-1α has been reported to play a crucial role in stem cell homing and recruitment to injured sites. However, no information is available about its role in periodontal tissues. The aim of this in vitro study was to investigate the effects of basic fibroblast growth factor (FGF-2) and transforming growth factor (TGF)-ß1 on SDF-1α expression in immortalized periodontal ligament (PDL) cells derived from deciduous teeth (SH9 cells). Real-time PCR and western blot analyses showed that SDF-1α mRNA expression in SH9 cells was markedly inhibited by FGF-2 treatment for 48 h. SU5402, which directly interacts with the catalytic domain of the FGF receptor 1 (FGFR1) and suppresses its phosphorylation, inhibited the FGF-2-related decrease in SDF-1α expression. These results suggest that FGF-2 signaling via the FGFR1 pathway inhibits SDF-1α expression. Conversely, SDF-1α expression in SH9 cells was increased by TGF-ß1 treatment for 12 h. Western blot analysis showed that this treatment induced Smad2/3 phosphorylation. A time-course experiment showed that SDF-1α expression levels reached a maximum 12 h after the TGF-ß1 treatment and returned to basal levels by 48 h. Real-time PCR analysis showed that Smad7 mRNA expression peaked by 6 h after TGF-ß1 treatment. Since Smad7 siRNA downregulated Smad7 expression by approximately 2.5-fold compared with the negative control siRNA, the induction of SDF-1α expression was prolonged. Furthermore, treatment of SH9 cells with TGF-ß1 for 12 h induced transwell migration of UE7T-13 cells, which are mesenchymal stem cells derived from human bone marrow. Therefore, SDF-1α may play an important role in stem and progenitor cell recruitment and homing to injured sites in the periodontal ligament, and regulation of SDF-1α expression may be a useful tool in cell-based therapy for periodontal tissue regeneration.

Fibroblast growth factor 2 inhibits the expression of stromal cell-derived factor 1α in periodontal ligament cells derived from human permanent teeth in vitro.

Although cells derived from periodontal ligament (PDL) tissue are reported to have stem cell-like activity and are speculated to play a crucial role for tissue healing and regeneration after injury or orthodontic treatment, mechanisms regulating their recruitment and activation remain unknown. Recently, stromal cell-derived factor 1α (SDF-1α) has been reported to be important for stem cell homing and recruitment to injured sites. The aim of this study was to evaluate whether fibroblast growth factor 2 (FGF-2) affects the expression of SDF-1α in PDL cells derived from human permanent teeth in vitro. Using real-time PCR, the expression of SDF-1α mRNA in PDL cells was inhibited by treatment with 10 ng/ml FGF-2. When PDL cells were treated with SU5402 (an inhibitor of FGF receptor 1) in combination with FGF-2, the FGF-2-reduced expression of SDF-1α was inhibited. In the presence of the JNK inhibitor SP600125, SDF-1α mRNA in PDL cells was not suppressed by the FGF-2 treatment. Western blot analysis also showed that SDF-1α production was suppressed by treatment with FGF-2, but it recovered with treatment by FGF-2 + SU5402. These findings suggest that SDF-1α from PDL cells plays an important role in the regeneration and homeostasis of periodontal tissues via the recruitment of stem cells.

Establishment of immortalized human periodontal ligament cells derived from deciduous teeth.

Although periodontal ligament (PDL) cells have previously been isolated from permanent teeth, they have not been isolated from deciduous teeth. Here, we used human telomerase reverse transcriptase (hTERT) induction to establish the first immortalized PDL cell lines derived from deciduous teeth. Cells were transfected with plasmids containing hTERT. Single-cell cloning was then performed using the limited dilution method. Reverse transcriptase polymerase chain reaction and stretch PCR were used to detect hTERT expression in the clones. In order to determine whether the clones could differentiate into osteoblasts, we stimulated the cells with ascorbic acid and ß-glycerophosphate. We success-fully obtained 3 single-cell clones, and named them single cell derived from human deciduous PDL (SH) 9, 10 and 11. All the SH cells showed hTERT expression and stable proliferation after >80 population doublings and expressed the marker genes of PDL cells, including scleraxis, periostin, cementum-derived protein 23, and tenomodulin. Although all the clones expressed osteoblastic markers, only the clones from the SH 9 cell line differentiated into osteoblastic cells. This is the first report of the immortalization of PDL cells derived from deciduous teeth. These cells could be useful in studies investigating the cellular mechanisms and regenerative processes of human PDL cells.

Effect of fibroblast growth factor-2 on periodontal ligament cells derived from human deciduous teeth in vitro.

A blood supply is crucial for tissue healing and regeneration. Periodontal ligament (PDL) tissue is situated between the tooth root and alveolar bone, and cells derived from PDL tissue are reported to have stem cell-like activity. This study aimed to evaluate the potential of PDL cells derived from deciduous teeth to express endothelial cell (EC)-specific markers. Using quantitative PCR, we investigated whether PDL cells derived from human deciduous teeth express mRNA for the EC-specific markers: vascular endothelialcadherin (VE-cadherin), vascular endothelial growth factor receptor 2 (VEGFR2) and CD31 upon treatment with 15 ng/ml heparin or 10 ng/ml fibroblast growth factor (FGF)-2 in vitro. Quantitative PCR showed that PDL cells expressed mRNA for the EC-specific markers, VE-cadherin and VEGFR2, when cultured in the presence of heparin alone or with FGF-2. By contrast, marked CD31 mRNA expression was induced only when PDL cells were cultured with both heparin and FGF-2. Western blot analysis showed that the CD31 protein was induced in PDL cells upon treatment with both heparin and FGF-2 for 3 weeks. PDL cells derived from deciduous teeth inducibly express EC-specific markers and thus have the potential to differentiate into a vascular cell lineage.