Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth.

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells. Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated. DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated. These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Bakopoulou A ，Leyhausen G ，Volk J ，Tsiftsoglou A ，Garefis P ，Koidis P ，Geurtsen W ... -  《-》

Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP).

The aim of this study was to compare the in vitro osteo/odontogenic differentiation potential of mesenchymal stem cells (MSCs) derived from the dental pulp (dental pulp stem cells - DPSCs) or the apical papilla (stem cells from the apical papilla - SCAP) of permanent developing teeth. DPSCs and SCAP cultures were established from impacted third molars of young healthy donors at the stage of root development. Cultures were analysed for stem cell markers, including STRO-1, CD146, CD34 and CD45 using flow cytometry. Cells were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4) and β-glycerophosphate. Cultures were analysed for morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers (dentine sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN, alkaline phosphatase-ALP), using immunocytochemistry and reverse transcriptase-polymerase chain reaction. All DPSCs and SCAP cultures were positive for STRO-1, CD146 and CD34, in percentages varying according to cell type and donor, but negative for CD45. Both types of MSCs displayed an active potential for cellular migration, organization and mineralization, producing 3D mineralized structures. These structures progressively expressed differentiation markers, including DSPP, BSP, OCN, ALP, having the characteristics of osteodentin. SCAP, however, showed a significantly higher proliferation rate and mineralization potential, which might be of significance for their use in bone/dental tissue engineering. This study provides evidence that different types of dental MSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source for osteo/odontogenic differentiation and biomineralization that could be further applied for stem cell-based clinical therapies.

Bakopoulou A ，Leyhausen G ，Volk J ，Tsiftsoglou A ，Garefis P ，Koidis P ，Geurtsen W ... -  《-》

Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells.

The aim of this study was to investigate the effects of resinous monomers on the odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP). Cultures were established from developing third molars of healthy donors aged 14-18 years-old and were extensively characterized for proliferation rate, colony forming unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then induced for odontogenic differentiation in the continuous presence of low concentrations (0.05-0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were evaluated. The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A down-regulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA. These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth.

Bakopoulou A ，Leyhausen G ，Volk J ，Koidis P ，Geurtsen W ... -  《-》

Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.

Bakopoulou A ，Leyhausen G ，Volk J ，Tsiftsoglou A ，Garefis P ，Koidis P ，Geurtsen W ... -  《-》

Coculture of dental pulp stem cells with endothelial cells enhances osteo-/odontogenic and angiogenic potential in vitro.

Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro. Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis. The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them. Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes.

Dissanayaka WL ，Zhan X ，Zhang C ，Hargreaves KM ，Jin L ，Tong EH ... -  《-》