Pro-inflammatory cytokine release and cell growth inhibition in primary human oral cells after exposure to endodontic sealer.

AIM: To assay the toxicity of the single-methacrylate-based sealer urethane dimethacrylate (UDMA) (EndoRez) in terms of cell growth and pro-inflammatory cytokines release, in expanded ex vivo human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPDLSCs), human gingival fibroblasts (hGFs) and human osteoblasts (hOSTs). METHODOLOGY: Dental pulp and periodontal ligament stem cells, osteoblasts and fibroblasts were derived from five young donors. After in vitro isolation, hDPSCs, hPDLSCs, hGFs and hOSTs were seeded to resin-based sealers for 24, 48, 72 h up to 1 week. The morphological features and the cell growth and the release of pro-inflammatory interleukin (IL)6, IL8, IL12 and tumour necrosis factor (TNF) α were analysed. Differences in cell growth and in interleukin secretion were analysed for statistical significance with two-way anova tests for multiple comparisons. RESULTS: Exposure to endodontic sealer based on UDMA resulted in a 50% decrease in survival oral cells at 24 h of incubation. No evident morphological changes were present in cell cultures examined. After 48 h, 72 h and 1-week culture time, a progressive cell growth was evident. A significant up-regulation of IL6, IL8, IL12 and TNFα cytokines in cells in contact with the dental sealer compared to the control was observed. CONCLUSION: In vitro, EndoRez interacted with primary human hDPSCs, hPDLSCs, hGFs and hOSTs causing damage to biological system evidenced through cell growth inhibition and up-regulation of IL6, IL8, IL12 and TNFα proinflammatory mediators.

Morphological analysis and interleukin release in human gingival fibroblasts seeded on different denture base acrylic resins.

The development of different types of materials with application in practice dentistry is an area of intense growth and research due to its importance in oral health. Among the diverse materials currently used in restoration or in dentures, the acrylic based resins have been widely employed. The release of toxic components and the changes on their physical and mechanical properties actually represent a goal of intensive research. In vivo analysis showed that the surface roughness of the acrylic resin represents a factor that could stimulate bacteria colonization and soft tissue inflammation. For this purpose, in this work, we have analyzed the cell response to acrylic based resins Ivoclar, Tokuso and Coldpack in basal conditions, unpolished, and after the polished procedure performed to reduce the surface roughness. Our in vitro results using human gingival fibroblasts (HGFs) showed a decrease of cell growth, evaluated by MTT assay starting at 24 h of incubation, in samples seeded on resins in basal conditions and after the polished procedure. This cell growth reduction was associated to evident morphological changes in unpolished materials. After 24 h of culture in presence of polished and unpolished resins a spontaneous release was present of pro-inflammatory cytokines such as interleukin-6 (IL-6) and -8 (IL-8), which was higher in unpolished resins, indicating that the polished procedure, minimizing the cytotoxicity process, could contribute to reduce the gingival inflammation processes.

Overexpression of interleukin-6 and -8, cell growth inhibition and morphological changes in 2-hydroxyethyl methacrylate-treated human dental pulp mesenchymal stem cells.

AIM: To evaluate morphological features, cell growth and interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion in expanded ex vivo human dental pulp mesenchymal stem cells (DP-MSCs) after exposure to 2-hydroxyethyl methacrylate (HEMA). METHODOLOGY: Dental pulp mesenchymal stem cells were derived from the dental pulps of 10 young donors. After in vitro isolation, DP-MSCs were treated with 3 and 5 mmol L(-1) HEMA, and after 24, 48 and 72 h of incubation, their morphological features, cell growth, IL-6 and IL-8 secretion were analysed. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons. RESULTS: Dental pulp mesenchymal stem cells revealed a decrease in cell growth with both treatments (P < 0.05), more evident at 5 mmol L(-1) . Microscopic analysis displayed extensive cytotoxic effects in treated cells, which lost their fibroblastoid features and became retracted, even roundish, with a large number of granules. An up-regulation of IL-6 and IL-8 in treated cells cytokines was evident (P < 0.05). CONCLUSIONS: 2-Hydroxyethyl methacrylate exhibited cytotoxicity, inhibited cell growth and induced morphological changes in cultured DP-MSCs. Moreover, in treated samples, an up-regulation of soluble mediators of inflammation such as IL-6 and IL-8 cytokines was found. The direct application of HEMA potentially induces an inflammation process that could be the starting point for toxic response and cell damage in DP-MSCs.

The cytotoxic effects of resin-based sealers on dental pulp stem cells.

AIM: To evaluate the effect of four current resin-based adhesives on expanded ex vivo human dental pulp mesenchymal stem cells (DP-MSCs). METHODOLOGY: Dental pulp mesenchymal stem cells were derived from dental pulps of ten donors. After in vitro isolation, dental pulp stem cells were analysed using flow cytometry. The immunophenotype of DP-MSCs disclosed the homogeneous expression of the mesenchymal-related antigens CD29, CD44, CD73, CD90, CD105, CD166. DP-MSCs were exposed to four different commercially available bonding systems (CMF Bond, Prime&Bond NT, Clearfil S(3) Bond, XP Bond), and after 24, 48 and 72 h of incubation the morphological features and the cell growth were analysed. Moreover, the cell viability was evaluated at the same times by MTT assay. Data were statistically analysed using a two-way anova and Holm-Sidak method (alpha set at 0.05). RESULTS: Significant differences were observed between the four groups when comparing DP-MSCs appearance. DP-MSCs survived and proliferated without inhibition in the presence of CMF Bond adhesive. On the contrary, microscopic evaluation of the other three groups revealed extensive cytotoxic effects from the dentine bonding agents. The MTT assay revealed no statistically significant differences in cell viability after 72 h between the control group and CMF Bond group. All the other experimental groups had statistically lower optical density values. CONCLUSIONS: CMF Bond adhesive allowed human dental pulp stem cells to survive and proliferate. All of the other dentine bonding agents had extensive cytotoxic effects.

The performance of human periodontal ligament mesenchymal stem cells on xenogenic biomaterials.

Mesenchymal stem cells from periodontal ligament (PDL-MSCs) hold great promise for bone regeneration. Most studies regarding the osteogenic differentiation of stem cells from periodontal tissue suggest that PDL cells may have many osteoblast-like properties, including the ability to form calcified nodules in vitro. This study investigated the morphological and histochemistry aspects of human PDL-MSCs, induced for osteogenic differentiation and seeded on a xenogenic porcine bone substitute in vitro, at different times of incubation. This biomaterial seems physically identical to human bone, and it has been reported to be osteoconductive. Our results indicated that the cells had a high affinity for the three-dimensional biomaterials; in fact, cellular proliferation and colonization was evident, and after 21 days the adherent cells started to detach themselves from the substrate, and at 30 days of incubation in differentiation medium, the cells completely lost the adhesion to the Petri's disk, englobing all bioparticles. In conclusion, the in vitro behaviour of PDL-MSCs and their relationship with three-dimensional scaffold biomaterials encourage in vivo investigations for their use in dental tissue regeneration.

Dental pulp stem cells bioadhesivity: evaluation on mineral-trioxide-aggregate.

Stem cells are undifferentiated cells that have the capacity to self-renew. They have been discovered in many adult tissues, including teeth. Dental Pulp Mesenchymal Stem Cells (DP-MSCs) are involved in dental repair by activation of growth factors, released after caries and have the ability to regenerate a dentin-pulp-like complex. The molecular/cellular research gives the possibility to grow new tissues and biological structures for clinical applications, providing cells for therapies including cell transplantation and tissue engineering. In this study DP-MSCs were derived from dental pulp of 10 donors. To evaluate material toxicity, after in vitro isolation, the cells were seeded on mineral trioxide aggregate (MTA). Initial light microscopy investigation of cells revealed no signs of cell death due to toxicity or infection, on the contrary the scaffolds supplied an excellent support for cell structures, the cells proliferated and adhered to substrate. Similar observation was seen in scanning electron microscopy, in particular the cells had proliferated and spread, covering a considerable part of the surface of the biomaterials investigated, with an elaborate form of attachment, in fact, the cells formed a continuous layer on the upper surface of the MTA. In conclusion, the aim of this study is to demonstrate that DP-MSCs combined with MTA could be a potential source for regenerative medicine, encouraging further study to evaluate the new dentin formation.

Changes of plasma membrane properties in a human pre-T cell line undergoing apoptosis.

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca(2+) concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.

Microbiological study and scanning electron microscopic analysis of root canal wall dentin following pumped Diodium Nd:YAG laser irradiation.

The capability of Nd:YAG laser in sterilizing root canals and the alterations of dentinal walls induced by laser treatment were investigated. Thirty root canals were infected by P. aeruginosa ATCC 27853 and thirty canals by A. naeslundii CH-12. Within each infection, 4 groups were selected on the basis of the treatment. Among them, 2 test groups (TGs) were treated by Nd:YAG laser at 15 Hz for 15 s, using 2 different settings: 1 Watt/70 Joule and 1.5 Watt/100 Joule, respectively (n = 10 each). The other 2 groups, used as controls (CGs), were: untreated (positive control, n = 5) and sterilized by 5.25% NaClO group (negative control, n = 5). Observations under scanning electron microscope (SEM) and quantitative bacterial counts were performed. These analyses were performed once per group after infections and treatments. Laser treatments significantly reduced the number of both bacteria. SEM investigation showed melting and crystallization of canal dentin over 1.5 W/100 J. Laser irradiation has a bactericidal effect but it does not completely sterilize the root canal as NaClO 5.25% solution does if the goal of treatment is also to avoid alterations of dentinal walls.

Isotachophoresis as a useful tool for monitoring neurological complications of acute leukaemia in children.

Cerebrospinal fluid proteins from 42 children with acute lymphoblastic leukaemia were analysed by isotachophoresis. The isotachopherograms of cerebrospinal fluid taken from patients undergoing central nervous system prophylaxis with neurological complications showed an increase of several peaks (albumin, prealbumin, and an unidentified peak), and changes in the globulin zone, compared with those from patients who had completed central nervous system prophylaxis for at least six months. The most striking finding was that these alterations were not associated with any other biochemical changes in the cerebrospinal fluid, as assayed by routine analysis. Isotachophoresis may be useful in the monitoring of therapy in children affected with acute lymphoblastic leukaemia.

In vitro effect of levamisole on oxygen consumption and survival of platelets.

Levamisole at a concentration of 10(-3)M inhibits the oxygen consumption of resting platelets, the thrombin induced burst and the platelet aggregation induced by ADP. At the concentration of 10(-7)M it exerts still an inhibitory activity of the thrombin induced burst, but it does not inhibit neither the basal oxygen consumption nor the platelet aggregation. At every tested concentration platelet survival in the presence of levamisole is comparable to that of controls. Levamisole moreover, together with theophylline and glucagon, shows a synergistic inhibiting influence toward the burst of oxygen consumption. Our data suggest that levamisole may act by producing an enzymatic block of cyclo-oxygenase.