Dental pulp inflammatory/immune response to a chitosan-enriched fibrin hydrogel in the pulpotomised rat incisor.

Current pulpotomy is limited in its ability to induce regeneration of the dental-pulp (DP) complex. Hydrogels are reported to be well-suited for tissue engineering and are unlikely to induce an inflammatory response that might damage the remaining tissue. The present study investigated the molecular and cellular actors in the early inflammatory/immune response and deciphered M1/M2 macrophage polarisation to a chitosan-enriched fibrin hydrogel in pulpotomised rat incisors. Both fibrin and fibrin-chitosan hydrogels induced a strong increase in interleukin-6 (IL-6) transcript in the DP when compared to the DP of untreated teeth. Gene expression of other inflammatory mediators was not significantly modified after 3 h. In the viable DP cell population, the percentage of leukocytes assessed by flow cytometry was similar to fibrin and fibrin-chitosan hydrogels after 1 d. In this leukocyte population, the proportion of granulocytes increased beneath both hydrogels whereas the antigen-presenting cell, myeloid dendritic cells, T cells and B cells decreased. The natural killer (NK) cell population was significantly decreased only in DPs from teeth treated with fibrin-chitosan hydrogel. Immunolabeling analysis of the DP/hydrogel interface showed accumulation of neutrophil granulocytes in contact with both hydrogels 1 d after treatment. The DP close to this granulocyte area contained M2 but no M1 macrophages. These data collectively demonstrated that fibrin-chitosan hydrogels induced an inflammatory/immune response similar to that of the fibrin hydrogel. The results confirmed the potential clinical use of fibrin-chitosan hydrogel as a new scaffold for vital-pulp therapies.

Psychometric validation of the French version of the Connor-Davidson Resilience Scale.

OBJECTIVES: Resilience defines the ability to face adversity with positive outcomes. Different scales, including the 25-item Connor-Davidson Resilience Scale (CDRISC), have been elaborated in order to evaluate resilience among various populations. The evaluation of resilience in French populations was impossible until CDRISC was translated into French. In the present work, we aim to validate a French version of CDRISC (f-CDRISC). METHODS: The survey was conducted at Nantes University. Both dental and medical students were eligible. The factor structure of f-CDRISC was determined and its replicability was tested on two sub-samples by exploratory factor analysis (EFA) and parallel analysis (PA). A third student sample was used for confirmatory factorial analysis (CFA). RESULTS: We collected 1210 responses. Four items did not reach acceptance thresholds for reliability and were discarded from the f-CDRISC. EFA and PA of the remaining 21 items highlighted a replicable 3-factor structure that was further confirmed by CFA. Resilience factors included "tolerance to negative affects", "tenacity" and "self-confidence". All factors displayed acceptable to good internal consistency. They were characterized by positive medium to strong correlations with the overall f-CDRISC Scale. Significant positive correlations were also observed between the resilience factors. CONCLUSION: The present work constitutes the first study devoted to a French adaptation of the CDRISC questionnaire. We present evidence showing that the f-CDRISC is a reliable tool for resilience evaluation in French speaking populations.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Study of the impacts of patient-educators on the course of basic sciences in dental studies.

Ever since 2006, Nantes University dental educators have started organising lectures led by the mother of a young patient suffering from ectodermic dysplasia (patient-educator) to help second-year students to better understand how important it is for their future dental work to better understand basic sciences. In this study, we have analysed this training experience on students' motivation. For this purpose, students were asked to complete questionnaires 10 days after the patient-educator's lecture (early assessment; n = 193) and 4 years later, during the last year of their dental studies (delayed assessment; n = 47). Moreover, 3 years after the first lecture, we analysed the ability of students to diagnose a mother carrying the ectodermic dysplasia genetic disorder, using a case-based learning exercise with a patient showing dental features similar to those exposed by the patient-educator (measure of knowledge; n = 42). Ten days after the lecture, the early assessment shows that all the students were interested in the lecture and 59% of the students declared being motivated to find out more about genetics whilst 54% declared the same thing about embryology courses. Moreover, 4 years later, 67% of the students remembered the patient-educator's lecture a little or very well. Three years after the course, 83% of the students diagnosed ectodermal dysplasia whilst studying the case-based example that listed typical dental phenotypes. In conclusion, this study shows that this original educational approach enhances dental students' motivation in learning basic sciences and that patient-educators could offer many benefits for students and patients.

Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells.

OBJECTIVE: Dental pathologies such as caries are the most prevalent disease worldwide with infectious and social complications. During the process of caries formation, the tooth is degraded and demineralization of enamel and dentine leads to the release of large amounts of inorganic phosphate (Pi) within dental tubuli. As Pi has been shown to induce apoptosis in skeletal cells, including osteoblasts and chondrocytes, we questioned whether high concentrations of Pi could affect odontoblast viability, proliferation and apoptosis. DESIGN: Using the odontoblast-like MO6-G3 cell line as a model, we used cell counting and MTS-based colorimetric assays to measure cell viability and proliferation. Apoptosis was assessed using Hoechst nuclei staining and detection of the early apoptotic markers annexin V and Apo2.7. RESULTS: We show for the first time that a high Pi concentration (7 mM) induced a decrease in odontoblast viability and proliferation together with a large increase in apoptosis. These effects were blunted in calcium-free medium, possibly due to the formation of calcium-phosphate crystals in the presence of high Pi concentrations. CONCLUSION: This study contributes to clarifying the effect of Pi on odontoblast viability and apoptosis, which may improve our understanding of the role of Pi during caries formation.

Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates.

Clinical translation of dendritic cell (DC)-based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol-DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow-derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol-DCs that were poor stimulators of T cells and actively inhibited T-cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti-inflammatory and immunosuppressive enzyme heme oxygenase-1 (HO-1) was up-regulated in Tol-DCs, compared to the immunogenic BMDCs. We demonstrated that HO-1 mediates the immunosuppressive properties of Tol-DCs in vitro (in NHPs and rats) and that HO-1 is involved in the in vivo tolerogenic effect of Tol-DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP Tol-DCs. Furthermore, we showed for the first time that HO-1 plays a role in the active inhibition of T-cell responses by rat and NHP Tol-DCs.

Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2.

Sodium-dependent phosphate cotransporters are key regulators of phosphate homeostasis and play a major role in mineralized tissues remodelling. However, factors influencing their expression remain under consideration. In our study, modulation of type III sodium-dependent phosphate cotransporters expression by inorganic phosphate (Pi) was investigated in the murine odontoblast-like cell line MO6-G3. Experiments were designed to determine the effects of phosphate release on dental cells during tooth decay. By real-time RT-PCR we demonstrated that Glvr-1 and -2 expressions are up-regulated by Pi. The increase in Glvr-1 and -2 expressions was correlated with ERK1/2 phosphorylation and calcium/phosphate crystals formation in cultured wells. Using calcium-free culture conditions or the specific inhibitor of ERK phosphorylation (UO126), we demonstrated that Pi effects on Glvr-1 and -2 up-regulation require the presence of calcium and involve ERK signalling pathways. This study contributes to give new insights in the control of Pi transport during carious diseases.

Culture medium modulates the behaviour of human dental pulp-derived cells: technical note.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Characterization of alpha-smooth muscle actin positive cells in mineralized human dental pulp cultures.

In response to injury, pulp precursor cells can differentiate into odontoblast-like cells that produce reparative dentine. In culture, pulp cells form mineralizing nodules, but the characteristics of the cells involved in this process are still not fully known. Human pulp cells for culture were obtained from coronal pulp isolated from non-erupted molars, and were maintained in RPMI 1640 medium supplemented with fetal calf serum. Nodules were forming in all human pulp primary cultures (HPPc) and human pulp subcultures observed until their fifth passage (HPSc<5). Mineralization of the nodules was confirmed by the presence of calcium and phosphate that were quantified by X-ray microanalysis. Specific immunolabeling revealed alpha-smooth muscle actin and vimentin in both HPPc and HPSc<5 cells. Cells positive for alpha-smooth muscle actin were either isolated or gathered together in the nodules. Under transmission electron microscopy, some cells in primary pulp cultures exhibited features typical of myofibroblasts or pericytes, such as stress fibers, fibronexus, indented nuclei and gap-junctions. These cells were frequently in close contact with mineral deposits. This work demonstrates for the first time the presence of pericytes or myofibroblasts in mineralized human pulp cultures, but further investigation is required to determine their origin, role and degree of differentiation.

Effects of hydroxyapatite particles on periodontal ligament fibroblast-like cell behavior.

Although hydroxyapatite (HA), a synthetic calcium phosphate, is used in restoring bone defects associated with periodontal diseases, its specific effect on the periodontal ligament fibroblast population during the regeneration process is unclear. To determine the cellular events occurring in the presence of HA, human periodontal ligament fibroblasts (HPLF) were isolated and maintained in culture. The specificity of the cells was evidenced by their morphology, deposition of extracellular matrix components, and alkaline phosphatase (ALP) activity (as a marker of osteoblastic differentiation of HPLF). Phase-contrast investigations revealed morphological alterations of cells in contact with HA particles. Transmission electron microscopy demonstrated the phagocytotic process of HPLF toward HA particles. Moreover, the presence of HA particles was significantly related to an increase in the protein synthesis activity and a decrease in the proliferation and ALP-specific activity of HPLF. These results provide new information on the phenotypic expression of HPLF, which is comparable to that of osteoblastic cells. A subpopulation of HPLF may be influenced by the presence of HA to undergo transient dedifferentiation prior to redifferentiating into osteoblasts. This process may be important as a means by which HA acts as an osteoconductive material. This experimental study improves our understanding of the cellular processes which occur during healing and regeneration of periodontal defects after implantation of biomaterials.

Comparative effect of calcium hydroxide and hydroxyapatite on the cellular activity of human pulp fibroblasts in vitro.

When, in vivo, calcium hydroxide [Ca(OH)2] or hydroxyapatite are used as dental pulp-capping agents, a reparative dentine bridge is observed. New hard tissue is formed directly on the hydroxyapatite, whereas a characteristic necrotic area appears under Ca(OH)2. The differing pulpal reactions to these two capping agents suggest differing cell responses. After isolation and selection of human pulp fibroblasts in vitro, the cells were characterized by their morphology, their high alkaline phosphatase specific activity, and their synthesis of type I and III collagens and fibronectin. They were then incubated in the presence of either hydroxyapatite (1 mg/ml) or Ca(OH)2 (0.8 mg/ml). With Ca(OH)2, the cells exhibited dramatical alterations in morphology, DNA synthesis, alkaline phosphatase activity and protein synthesis, in accordance with the necrosis observed in vivo. With hydroxyapatite, phagocytic activity of pulpal fibroblasts toward hydroxyapatite particles (< 10 microns) was seen. As a consequence, DNA synthesis was affected. This inhibitory effect was not due to cell damage, as demonstrated by increased [3H]-proline and [3H]-leucine incorporation by the cells. There was also an inhibitory effect of hydroxyapatite on alkaline phosphatase activity, suggesting that the pulp fibroblasts were not in a differentiation stage. In conclusion, compared to the effects of Ca(OH)2 on human pulp fibroblasts, these data are consistent with the biocompatibility of hydroxyapatite previously described in vivo and testify to the occurrence of a biological response elicited by this synthetic biomaterial.

Cellular activity of osteoblasts in the presence of hydroxyapatite: an in vitro experiment.

The use of synthetic calcium phosphate as bone substitute calls for the knowledge of the influence on adjacent cells. Effects on monocytes, macrophages, synovial cells and fibroblasts have been largely described in vivo and in vitro but few data are available as concerns osteoblast responses. The present experiments tested the activity of MC3T3-E1, ROS 17/2.8 and mouse calvaria cells cultured in the presence of hydroxyapatite powders. The three osteoblast-like cells were shown to phagocytoze the calcium phosphate particles. As a consequence, they exhibit reduced cell growth and alkaline phosphatase activity. This response was different when compared with other cell types. The osteogenetic function of osteoblastic cells could be involved in these specific effects of hydroxyapatite.