Differential inflammatory response of dental pulp explants and fibroblasts to saliva

Abstract AIM: To investigate the inflammatory response of dental pulp fibroblasts and the respective explants to whole saliva. METHODOLOGY: Explants from human and porcine dental pulp tissue and isolated dental pulp fibroblasts were used to investigate the inflammatory response to sterile saliva. Cytokine and chemokine expression was assessed by RT-PCR. Western blot analysis and pharmacologic inhibitors were used to determine the involvement of signalling pathways. RESULTS: Dental pulp explants of human and porcine origin exposed to human saliva exhibited no major changes of IL-6 and IL-8 mRNA expression (P > 0.05). In contrast, isolated porcine and human dental pulp fibroblasts, when stimulated with human saliva, exhibited a vastly increased expression of IL-6 and IL-8 mRNA (P < 0.05). In pulp fibroblasts, saliva also increased the expression of other cytokines and chemokines via activation of NFkappaB, ERK and p38 signalling. Notably, a significantly reduced inflammatory response was elicited when pulp fibroblasts were transiently exposed to saliva. CONCLUSIONS: Saliva has a potential impact on inflammation of dental pulp fibroblasts in vitro but not when cells are embedded in the intrinsic extracellular matrix of the explant tissue.

Effects of Prolyl Hydroxylase Inhibitor L-mimosine on Dental Pulp in the Presence of Advanced Glycation End Products

INTRODUCTION Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp-derived cells (DPCs) and tooth slice organ cultures. METHODS In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology. RESULTS In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed. CONCLUSIONS Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

Temperature changes in dental pulp associated with use of power grinding equipment on equine teeth

Objective To quantify the temperature changes in the dental pulp associated with equine dental procedures using power grinding equipment. Design A matrix experimental design with replication on the same sample was followed to allow the following independent variables to be assessed: horse age (young or old), tooth type (premolar or molar), powered grinding instrument (rotating disc or die grinder), grinding time (15 or 20 seconds) and the presence or absence of water coolant. Procedure Sound premolar and molar teeth from a 6-year-old horse and a 15-year-old horse, which had been removed postmortem, were sectioned parallel to the occlusal plane to allow placement of a miniature thermocouple at the level of the dental pulp. The maximum temperature increase, the time taken to reach this maximum and the cooling time were measured (n=10 in each study). The teeth were placed in a vice and the instrument used on the tooth as per clinical situation. Results Significant differences were recorded for horse age (P < 0.001), instrument type (P < 0.001), grinding time (P < 0.001) and presence or absence of coolant (P < 0.001). There was no significant difference for tooth type. Conclusion Thermal insult to the dental pulp from the use of power instruments poses a significant risk to the tooth. This risk can be reduced or eliminated by appropriate selection of treatment time and by the use of water irrigation as a coolant. The increased dentine thickness in older horses appears to mitigate against thermal injury from frictional heat.

In vitro assessment of pulp chamber temperature of different teeth submitted to dental bleaching associated with LED/laser and halogen lamp appliances

This study sought to assess the pulp chamber temperature in different groups of human teeth that had been bleached using hydrogen peroxide gel activated with halogen lamps or hybrid LED/laser appliances. Four groups of ten teeth (maxillary central incisors, mandibular incisors, mandibular canines, and maxillary canines) were used. A digital thermometer with a K-type thermocouple was placed inside pulp chambers that had been filled with thermal paste. A 35% hydrogen peroxide-based red bleaching gel was applied to all teeth and photocured for a total of three minutes and 20 seconds (five activations of 40 seconds each), using light from an LED/laser device and a halogen lamp. The temperatures were gauged every 40 seconds and the data were analyzed by three-way ANOVA, followed by Tukey's test. Regardless of the light source, statistically significant differences were observed between the groups of teeth. The mean temperature values (±SD) were highest for maxillary central incisors and lowest for mandibular canines. The halogen lamp appliance produced more pulp chamber heating than the LED/laser appliance. The increase in irradiation time led to a significant increase in temperature.

Influence of the coloring agent concentration on bleaching gel and pulp chamber temperatures during dental bleaching

This study evaluated the Influence of the coloring agent concentration on the temperature of the gel layer and pulp chamber during dental bleaching with an LED/laser light source. Ten human incisors and a digital thermometer with K-type thermocouples were used. Using a high-speed spherical diamond bur, endodontic access was gained through openings on the lingual faces until pulp chamber was exposed. One end of the thermocouple was placed on the labial surface (immersed in bleaching gel) and the other end in the pulp chamber. The same 10 specimens were used in the 12 groups, according to the type and concentration of bleaching gel. Each bleaching gel was used in four different concentrations: manipulated without coloring, with normal quantity recommended by the manufacturer, with double the recommended amount of coloring, and with triple the recommended amount of coloring. The temperature rise was measured every 30 seconds for three minutes with a K-type thermocouple. The data were analyzed by ANOVA to examine the concentration and type of bleaching gel. This test was followed by Tukey's test, which was performed Independently for the gel at the labial surface and the pulp chamber (a = 5%). For both surfaces, values of p = 0.00 were obtained for all factors and for the Interaction between them. The varying concentrations of coloring agent produced statistically significant differences in terms of temperature increase for both the gel layer and the pulp chamber during activation.

Diabetes triggers the loss of tooth structure associated to radiographical and histological dental changes and its evolution to progressive pulp and periapical lesions in rats

The aim of this study was to evaluate the putative influence of diabetes without metabolic control in the loss of tooth structure as well as histological changes in dentin and pulp tissue in rats. Diabetes was induced in Wistar rats (n=25) by intravenous administration of alloxan (42mg/kg). Diabetic and non-diabetic control rats were evaluated at 1, 3, 6, 9 and 12 months of follow-up. In order to evaluate the presence and progression of dental caries and periapical lesions, hemimandibles were removed and submitted to radiographical, histological, and morphometrical procedures. Dental caries were detected after radiographical and histological evaluations in diabetic group from the third month of diabetes onset, increasing gradually in frequency and severity in periods. Diabetic rats dental pulps also presented significant reduction in volume density of collagen fibers and fibroblasts at third month, parallel with a trend towards the increase in inflammatory cells volume density. Diabetic rats presented a generalized pulp tissue necrosis after 6 months of diabetes induction. Moreover, periapical lesions were not detected in control group, while these lesions were observed in all rats after 3, 6, 9, and 12 months of diabetes induction. Uncontrolled diabetes seems to trigger the loss of tooth structure, associated to histological dental changes and mediates its evolution to progressive severe pulp and periapical lesions in rats. Therefore, diabetes may be considered a very important risk factor regarding alterations in dental pulp, development of dental caries, and periapical lesions.

NPY Y1 and VPAC1 receptor expression in human dental pulps

Neuropeptides such as neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) have been shown by our research group to be present in human dental pulp tissue. Neuropeptides cannot cross cell membranes and therefore to exert their biological effects they must bind to selected receptors on the surface of target cell membranes. However, the expression of receptor proteins for NPY and/or VIP have yet to be reported in human pulp tissue. The presence of neuropeptide receptors can be conveniently determined by Western blotting using specific anti-receptor antibodies. Objectives: The aim of this work was to identify the presence of the NPY Y1 receptor and the VIP receptor VPAC1 in human dental pulp tissue from both intact and carious teeth using Western blotting. Methods: Pulp tissue was collected from both intact and carious teeth and membrane preparations from these tissues were then subject to sodium dodecyl sulphate gel electrophoresis (SDS-PAGE), transferred to nitrocellulose and probed with specific antibodies to either the NPY Y1 receptor or the VPAC1 receptor. Results: Individual Western blotting experiments revealed the presence of immunoreactive bands corresponding to the known molecular weights of the NPY Y1 and VPAC1 receptor proteins in both intact and carious pulp samples. Conclusions: Demonstration of the presence of NPY Y1 and VPAC1 receptor protein expression in pulpal tissue from intact and carious teeth provides further support for the roles of these neuropeptides in pulpal health and disease.

Vasoactive Intestinal Polypeptide (VIP) in Human Dental Pulps

Pulpal innervation is not exclusively sensory and there are potential roles for other neuropeptides such as vasoactive intestinal polypeptide (VIP) in pulpal health and disease. In the systemic circulation VIP relaxes vascular smooth muscles leading to vasodilatation. It has been shown that VIP fibres are associated with pulpal blood vessels and therefore VIP may mediate vasoactivity in the dental pulp. A growing body of evidence has now demonstrated that an additional major physiological role of VIP is to act as a survival factor. In order to gain a better understanding of the role of neuropeptides in the caries process it is of interest to specifically examine a role for VIP. Objectives: The aim of the present study was to determine the levels of VIP in carious (moderately carious and grossly carious) compared with non-carious teeth. Methods: A total of 68 teeth were included in the study (22 non-carious, 20 moderately carious and 26 grossly carious). VIP was measured in all samples using a sensitive and specific radioimmunoassay. Results: The mean concentration of VIP in the pulps of non-carious teeth was 7.69 ng/g (9.41 SD) compared to 14.93 ng/g (15.58 SD) in carious teeth. Pair-wise comparisons of VIP levels using Tukey’s test showed statistically significant differences in VIP expression between non-carious and moderately carious teeth (p=0.002) and between moderately and grossly carious teeth, (p=0.002). Conclusion: The significantly increased levels of VIP in moderately carious pulps compared with either non-carious or grossly carious pulps may suggest a role for VIP as a protective or survival factor.

Radioimmunoassay Quantitation of Neuropeptide Y (NPY) in Human Dental Pulps

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundantly expressed in both the central and peripheral nervous systems. NPY has previously been shown to be present in human dental pulp although its exact role in pulpal health and disease remains to be fully elucidated. In addition to serving a neurotransmitter role, NPY may also have a role in modulating the pulpal response to injury and inflammation. Indeed NPY is known to be a potent vasoconstrictor in a range of tissues. Recent work by our research group has demonstrated changes in sensory neuropeptide levels measured by radioimmunoassay (RIA) in healthy and carious teeth. In addition to elevated levels of sensory neuropeptides, it is also possible that the carious process is associated with increased levels of autonomic neuropeptides such as NPY. Objectives: The aim of the present study was to undertake a comprehensive quantitative RIA analysis of NPY expression in human dental pulps from carious and non-carious teeth. Methods: A total of 22 non-carious and 46 carious teeth were included in the study. NPY was measured in all samples using RIA. Briefly, the RIA system consisted of a total volume of 400 ul, comprising 100 ul anti-NPY antibody (Peninsula Laboratories), 200 ul human NPY synthetic standard or pulp sample, and 100 ul of 125I-labelled NPY as radioactive tracer. Results: The mean concentration of NPY in non-carious teeth was found to be 4.28 ng/g (4.34 SD) compared to 9.57 ng/g (9.39 SD) in carious teeth. Using ANOVA the difference in NPY levels between the non-carious group and the carious group was found to be statistically significant (p= 0.003). Conclusion: The significant increase in the levels of NPY in carious dental pulps reported in this study provides evidence for a role for NPY in the pulpal response to caries.

Influência da espessura do substrato dental sobre a eficácia clareadora e citotoxicidade de diferentes protocolos de clareamento

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro analysis of human tooth pulp chamber temperature after low-intensity laser therapy at different power outputs

In vitro studies have provided conflicting evidence of temperature changes in the tooth pulp chamber after low-level laser irradiation of the tooth surface. The present study was an in vitro evaluation of temperature increases in the human tooth pulp chamber after diode laser irradiation (GaAlAs, lambda = 808 nm) using different power densities. Twelve human teeth (three incisors, three canines, three premolars and three molars) were sectioned in the cervical third of the root and enlarged for the introduction of a thermocouple into the pulp chamber. The teeth were irradiated with 417 mW, 207 mW and 78 mW power outputs for 30 s on the vestibular surface approximately 2 mm from the cervical line of the crown. The highest average increase in temperature (5.6A degrees C) was observed in incisors irradiated with 417 mW. None of the teeth (incisors, canines, premolars or molars) irradiated with 207 mW showed temperature increases higher than 5.5A degrees C that could potentially be harmful to pulp tissue. Teeth irradiated with 78 mW showed lower temperature increases. The study showed that diode laser irradiation with a wavelength of 808 nm at 417 mW power output increased the pulp chamber temperature of certain groups of teeth, especially incisors and premolars, to critical threshold values for the dental pulp (5.5A degrees C). Thus, this study serves as a warning to clinicians that ""more"" is not necessarily ""better"".

Cytotoxicity and biocompatibility of direct and indirect pulp capping materials

There are several studies about the cytotoxic effects of dental materials in contact with the pulp tissue, such as calcium hydroxide (CH), adhesive systems, resin composite and glass ionomer cements. The aim of this review article was to summarize and discuss the cytotoxicity and biocompatibility of materials used for protection of the dentin-pulp complex, some components of resin composites and adhesive systems when placed in direct or indirect contact with the pulp tissue. A large number of dental materials present cytotoxic effects when applied close or directly to the pulp, and the only material that seems to stimulate early pulp repair and dentin hard tissue barrier formation is CH.

Response of the pulp of dogs to capping with mineral trioxide aggregate or a calcium hydroxide cement

This study was conducted to observe the response of dogs' dental pulp to mineral trioxide aggregate (MTA) and a calcium hydroxide cement when used as pulp capping materials. After the pulps of 30 teeth were exposed, they were capped with either MTA or a calcium hydroxide cement. Histological analysis was performed 2 months after treatment. Results showed a healing process with complete tubular dentin bridge formation and no inflammation in any of the pulps capped with MTA. on the other hand, only five specimens from the calcium hydroxide cement group formed a complete dentin bridge. In this experimental group, pulp inflammation was observed in all but three cases. In conclusion, MTA exhibited better results than the calcium hydroxide cement for the capping of the pulp in dogs.

Penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique

This study evaluated the pulp chamber penetration of peroxide bleaching agent in human and bovine teeth after office bleach technique. All the teeth were sectioned 3 mm apical of the cement-enamel junction and were divided into 2 groups, A (70 third human molars) and B (70 bovine lateral incisors), that were subdivided into A1 and B1 restored by using composite resin, A2 and B2 by using glass ionomer cement, and A3 and B3 by using resin-modified glass ionomer cement; A4, A5, B4, and B5 were not restored. Acetate buffer was placed in the pulp chamber, and the bleaching agent was applied for 40 minutes as follows: A1-A4 and B1-B4, 38% hydrogen peroxide exposure and A5 and B5, immersion into distilled water. The buffer solution was transferred to a glass tube in which leuco crystal violet and horseradish peroxidase were added, producing a blue solution. The optical density of the blue solution was determined by spectrophotometer and converted into microgram equivalents of hydrogen peroxide. Data were submitted to analysis of variance and Dunnett, Kruskal-Wallis, and Tukey tests (5%). A higher level of hydrogen peroxide penetrated into the pulp chamber in resin-modified glass ionomer cements in bovine (0.79 +/- 0.61 mu g) and human (2.27 +/- 0.41 mu g) groups. The bleaching agent penetration into the pulp chamber was higher in human teeth for any experimental situation. The penetration of the hydrogen peroxide depends on restorative materials, and under the conditions of this study human teeth are more susceptible to penetration of bleaching agent into the pulp chamber than bovine teeth.