Substance P/calcitonin gene-related peptide and their receptors expression in human periodontal ligament after root canal preparation with five different systems.

AIM: The purpose of this study was to quantify the effect of five different root canal preparation instruments on Substance P (SP), Calcitonin gene-related peptide (CGRP) and their receptors expression in healthy human periodontal ligament. METHODOLOGY: STROBE guidelines were used to design a study using 60 periodontal ligament samples obtained from healthy lower premolars where extraction was indicated for orthodontic reasons. Prior to extraction 40 of these premolars were equally divided into four groups and root canals were prepared using different systems: Mtwo, Reciproc Blue, HyFlex EDM and Plex-V. Ten premolars were prepared with hand files and served as a positive control group. The remaining 10 premolars where extracted without treatment and served as a negative control group. All periodontal ligament samples were processed to measure the expression of SP, CGRP and their receptors by radioimmunoassay. Kruskal-Wallis and Duncan tests were performed to determine statistically significant differences between the groups for each variable. RESULTS: Greater expression of all the peptides measured were found in the hand-file preparation group, followed by the Reciproc Blue, Mtwo, HyFlex EDM and Plex-V groups. The lower SP, CGRP and their receptors values were for the intact teeth control group. Kruskal-Wallis test showed statistically significant differences amongst groups (p < .001). Dunn post-hoc tests showed statistically significant differences in SP, CGRP and their receptors expression between the intact teeth and the hand-file and Reciproc Blue groups. Hand-file group showed significant differences with the other groups, except with Reciproc Blue, where no differences were observed in any of the peptides measured. Finally, no differences were observed between Plex-V and HyFlex in any of the peptides measured. CONCLUSIONS: Root canal preparation with hand files and Reciproc Blue generates the highest expression of SP, CGRP, NK1 and CGRP1R in human periodontal ligament, whilst Plex-V and HyFlex maintain the basal expression of neuropeptides and their receptors. Mtwo showed intermediate results between Reciproc Blue and HyFlex.

The effect of orthodontic forces on calcitonin gene-related peptide (CGRP) expression in the human periodontal ligament and its relationship with the human dental pulp.

Background: The purpose of this study was to quantify the effect of moderate and severe orthodontic forces on Calcitonin gene-related peptide (CGRP) expression in the healthy human periodontal ligament (PDL) and its possible relationship with the human dental pulp. Material and Methods: Ninety human periodontal ligament samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Prior to extraction, teeth were divided in 3 groups of 30 samples each: I) Untreated teeth control group; II) Moderate force group: A 56 g force was applied to the premolars for 24 hours; and III) Severe force group: A 224 g force was applied to the premolars for 7 days. All periodontal ligament samples were processed and CGRP was measured by radioimmunoassay. Results: Greater CGRP expression was found in the severe force group, followed by the moderate force group. The lower CGRP values were for the untreated teeth. Kruskal-Wallis test showed statistically significant differences between groups (p<0.001). LSD post hoc tests showed statistically significant differences in CGRP expression between the untreated teeth and the severe forces group (p<0.001). Differences between the moderate and severe force groups were statistically significant (p<0.001). There was no statistically significant differences between the untreated teeth and the moderate forces group (p<0.261). Conclusions: CGRP expression in human periodontal ligament increases when teeth are submitted to severe orthodontic forces. This elevated expression of CGRP, which is proportional to the applied force, may affect the way the dental pulp responds to different stimuli from the orthodontic forces. Key words:Calcitonin gene-related peptide, orthodontic force, human periodontal ligament, neurogenic inflammation.

Expression of substance P, calcitonin gene-related peptide and vascular endothelial growth factor in human dental pulp under different clinical stimuli.

BACKGROUND: The aim of this study was to measure the dental pulp inflammatory response through neuropeptides (SP and CGRP) as a response to occlusal trauma, orthodontic movements and a combination of both, as well as the angiogenic defense mechanism through VEGF expression, which could be the initial step to mineralized tissue formation. METHODS: Forty human dental pulp samples were collected from healthy first premolars with extraction indicated due to orthodontic reasons from a sample of 20 patients. Patients were divided into four groups with 10 premolars each (1 mandibular and 1 maxillary premolar from each patient): healthy pulp control group, occlusal trauma group, moderate orthodontic forces group; and occlusal trauma plus moderate orthodontic forces group. Stimuli were applied for 24 h before tooth extraction in all experimental groups. All samples were processed, and SP, CGRP, and VEGF were measured by radioimmunoassay. The Kruskal-Wallis test was performed to assess significant differences among groups and Mann-Whitney's U post hoc pairwise comparisons were also performed. RESULTS: The highest increase in SP, CGRP, and VEGF expressions was found in the occlusal trauma plus orthodontic forces group, followed by the moderate orthodontic forces, the occlusal trauma and the control groups, with statistically significant differences between all groups for each of the 3 peptides analyzed (Kruskal-Wallis p < 0.001). All possible pairwise post-hoc comparisons were also significant for each peptide analyzed (Mann-Whitney's U p < 0.001). CONCLUSION: SP, CGRP, and VEGF expressions significantly increase in human dental pulps when stimulated by occlusal trauma combined with moderate orthodontic forces, as compared with these two stimuli applied independently. Name of the registry: Importance of Neurogenic Inflammation in the Angiogenic Response of the Dental Pulp as a Defensive Response. TRIAL REGISTRATION NUMBER: NCT03804034. Date of registration: 01/15/2019 Retrospectively registered. URL of trial registry record: https://clinicaltrials.gov/ct2/show/NCT03804034?term=NCT03804034&draw=2&rank=1 .

In vivo efficacy of three different endodontic irrigation systems for irrigant delivery to working length of mesial canals of mandibular molars.

INTRODUCTION: Many in vitro studies have debated over the ability of different irrigant delivery and/or agitation systems to reach the apical third of curved root canals; however, little is known about irrigant penetration in vivo. Therefore, the purpose of this study was to compare the efficacy of the conventional endodontic irrigation needle, passive ultrasonic irrigation (PUI), and a negative pressure system for irrigant delivery to working length (WL) of mesial canals of mandibular molars. METHODS: Thirty mesial canals of 30 vital mandibular first or second molars were randomly assigned into 3 groups (n = 10): (1) Monoject syringe with 27-gauge needle; (2) PUI with IrriSafe tip; and (3) EndoVac system. All canals were treated following the same preparation protocol to size 35/0.04 by using 5.25% NaOCl as irrigant during preparation procedure. Before obturation, canals were irrigated with 1 mL of a radiopaque solution by using the assigned irrigation system, and a digital radiograph was taken by using a parallel technique. With the aid of image editing software the distance between WL and maximum irrigant penetration was measured. RESULTS: Mean distances for Monoject, PUI, and EndoVac groups were 1.51 mm, 0.21 mm, and 0.42 mm, respectively. Analysis of variance test showed statistically significant differences between groups (P < .001). Tukey honestly significant difference test showed statistically significant differences between the Monoject group and the other 2 groups (P < .001) but no significant differences between PUI and EndoVac groups (P = .06). CONCLUSIONS: PUI and EndoVac are more effective than the conventional endodontic needle in delivering irrigant to WL of root canals.

The effect of orthodontic forces on calcitonin gene-related peptide expression in human dental pulp.

INTRODUCTION: The purpose of this study was to quantify the effect of moderate and severe orthodontic forces on calcitonin gene-related peptide (CGRP) expression in healthy human dental pulp. METHODS: Thirty human dental pulp samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, teeth were divided into 3 groups of 10 premolars each: (1) the control group: healthy premolars without application of orthodontic forces; (2) the moderate force group: a 56-g force was applied to the premolars for 24 hours; and (3) the severe force group: a 224-g force was applied to the premolars for 24 hours. All dental pulp samples were processed, and CGRP was measured by radioimmunoassay. RESULTS: Greater CGRP expression was found in the severe force group followed by the moderate force group. The lower CGRP values were for the control group. The Kruskal-Wallis test showed statistically significant differences between groups (P < .0001). Least significant difference (LSD) post hoc tests showed statistically significant differences in CGRP expression between the control group and the severe force group (P < .0001) but not with the moderate force group (P = .06). Differences between the moderate and severe force groups were statistically significant (P < .0001). CONCLUSIONS: CGRP expression in human dental pulp increases when teeth are submitted to severe orthodontic forces.

Effect of experimentally induced occlusal trauma on substance p expression in human dental pulp and periodontal ligament.

INTRODUCTION: The purpose of this study was to quantify the effect of occlusal trauma experimentally induced with occlusal interferences on substance P (SP) expression in healthy human dental pulp and periodontal ligament. METHODS: Twenty-eight human dental pulp and periodontal ligament samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, occlusal trauma was induced with experimental occlusal interferences in half of these premolars by placing a resin block over their occlusal surface and submitting patients to chew gum for 30 minutes. The remaining healthy premolars were extracted without occlusal trauma and served as a control group. All dental pulp and periodontal ligament samples were processed, and SP was measured by radioimmunoassay. RESULTS: There was 45% and 120% greater SP expression in dental pulp and periodontal ligament, respectively, of teeth with experimentally induced occlusal trauma. Paired t test showed statistically significant differences for both human dental pulp and periodontal ligament (P = .02 and P < .001, respectively) of teeth submitted to occlusal trauma when compared with control group values. CONCLUSIONS: SP expression in human dental pulp and periodontal ligament increases when teeth are submitted to occlusal trauma experimentally induced with occlusal interferences.

The effect of three different rotary instrumentation systems on substance P and calcitonin gene-related peptide expression in human periodontal ligament.

INTRODUCTION: The purpose of this study was to quantify the effect of three different rotary root canal preparation systems on substance P and calcitonin gene-related peptide expression in healthy human periodontal ligament. METHODS: Fifty periodontal ligament samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, 40 of these premolars were equally divided into four groups, and root canals were prepared using four different systems: the ProTaper Universal rotary system, the RaCe rotary system, the Mtwo rotary system, and the hand instrumentation technique. The remaining 10 healthy premolars that were extracted without treatment served as a negative control group. All periodontal ligament samples were processed, and SP and CGRP were measured by radioimmunoassay. RESULTS: Greater SP and CGRP expression were found in the ProTaper Universal group followed by the hand instrumentation group, the RaCe, and the Mtwo groups. The lower SP and CGRP values were for the negative control group. The Kruskal-Wallis test showed statistically significant differences between groups (p < 0.0001). Post hoc Least Significant Difference (LSD) tests showed statistically significant differences in SP and CGRP expression between the negative control group and all the other groups except the Mtwo group. Hand instrumentation also showed statistically significant differences with all the other groups, except the ProTaper Universal group. Differences between the three rotary systems were also statistically significant. CONCLUSION: SP and CGRP expression in periodontal ligament increases when teeth are prepared with ProTaper Universal and RaCe rotary instrumentation systems as well as with hand instrumentation. Mtwo maintains SP and CGRP levels.

The effect of different vasoconstrictors and local anesthetic solutions on substance P expression in human dental pulp.

The purpose of this study was to quantify the effect of the infiltration injection of different vasoconstrictor and anesthetic solutions on substance P (SP) expression in healthy human dental pulp. Thirty pulp samples were obtained from healthy upper premolars in which extraction was indicated for orthodontic reasons and were randomly assigned into three groups of 10 samples each: 2% lidocaine with 1:80,000 epinephrine (Lido group), 3% Prilocaine with 1:200000 felypressin (Prilo group); and 4% Prilocaine without vasoconstrictor (Prilo-no-VC group). All teeth were extracted 10 minutes after anesthetic application. Pulp samples were processed and SP was measured by radioimmunoassay. SP expression for the Lido, Prilo, and Prilo-no-VC groups were 616.49, 663.76, and 760.79 pmol/mg pulp tissue, respectively. Analysis of variance showed statistically significant differences between groups (p = 0.001). Tukey Honestly Significant Difference (HSD) post hoc tests showed significant statistical differences between the Prilo-no-VC group and the Lido group (p < 0.01) and between the Prilo-no-VC group and the Prilo group (p < 0.05). It can be concluded that infiltration injection of local anesthetics with vasoconstrictor attenuate SP expression in human dental pulp.

The effect of tooth bleaching on substance P expression in human dental pulp.

The purpose of this study was to quantify the effect of tooth bleaching on substance P (SP) expression in healthy human dental pulp. Forty pulp samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Thirty of these premolars were assigned into three different tooth-bleaching protocols: group 1 (n = 10): Opalescence Xtra Boost (Ultradent Products, South Jordan, UT) (38% H(2)O(2)) for 15 minutes; group 2 (n = 10): Lase Peroxide (DMC, Brazil) (35% H(2)O(2)) activated with infrared laser diode (Biolux; BioArt, Brazil) for 3 minutes, and group 3 (n = 10): Zoom! Whitening System (Discuss Dental, Culver City, CA) (25% H(2)O(2)) light activated for 20 minutes. The remaining 10 healthy premolars serve as a control group. Teeth were anesthetized immediately after bleaching and were extracted 10 minutes later. All pulp samples were processed and SP was measured by radioimmunoassay. Greater SP expression was found in the Zoom! Whitening System, followed by the Lase Peroxide group, Opalescence Xtra Boost, and the lower SP values were for the control group. Analysis of variance showed statistically significant differences between groups (p = 0.0001). Tukey HSD post hoc tests showed significant differences in the light (p < 0.01) and laser (p < 0.05) activated bleaching systems when compared with control values. It can be concluded that light- and laser-activated tooth-bleaching systems increase SP expression in human dental pulp significantly higher than normal values.

Calcitonin gene-related peptide receptor expression in alternatively activated monocytes/macrophages during irreversible pulpitis.

The purpose of this study was to quantify the percentage and the mean fluorescence intensity of viable alternatively activated monocytes/macrophages (AAMø) CD163+ positive for calcitonin gene-related peptide receptor (CGRPr) within the total AAMø population in human dental pulp. Pulp tissue samples were collected from teeth with a clinical diagnosis of irreversible pulpitis (n = 13), pulps with induced inflammation (n = 13), and normal pulps (n = 13). All samples were labeled to identify positive cells for CGRPr and CD163 using a flow cytometry assay. Results demonstrated that a high percentage of total viable AAMø CD163+ expressed CGRPr on their membranes (72.12% in healthy pulp, 62.20% in irreversible pulpitis, and 58.01% in induced pulpitis). Significant differences were found between mean AAMø CD163+ fluorescence for CGRPr according to pulp condition, being greater in irreversible pulpitis. It can be concluded that AAMø CD163+ are expressed during normal and inflammatory processes, supporting the hypothesis that they could exercise an anti-inflammatory action that could be controlled by CGRP signaling after its binding.

Neuropeptides in dental pulp: the silent protagonists.

Dental pulp is a soft mesenchymal tissue densely innervated by afferent (sensory) fibers, sympathetic fibers, and parasympathetic fibers. This complexity in pulp innervation has motivated numerous investigations regarding how these 3 major neuronal systems regulate pulp physiology and pathology. Most of this research is focused on neuropeptides and their role in regulating pulpal blood flow and the development of neurogenic inflammation. These neuropeptides include substance P, calcitonin gene-related peptide, neurokinin A, neuropeptide Y, and vasoactive intestinal polypeptide among others. The purpose of this article is to review recent advances in neuropeptide research on dental pulp, including their role in pulp physiology, their release in response to common dental procedures, and their plasticity in response to extensive pulp and dentin injuries. Special attention will be given to neuropeptide interactions with pulp and immune cells via receptors, including studies regarding receptor identification, characterization, mechanisms of action, and their effects in the development of neurogenic inflammation leading to pulp necrosis. Their role in the growth and expansion of periapical lesions will also be discussed. Because centrally released neuropeptides are involved in the development of dental pain, the pain mechanisms of the pulpodentin complex and the effectiveness of present and future pharmacologic therapies for the control of dental pain will be reviewed, including receptor antagonists currently under research. Finally, potential clinical therapies will be proposed, particularly aimed to manipulate neuropeptide expression or blocking their receptors, to modulate a variety of biologic mechanisms, which preliminary results have shown optimistic results.

Variation in the expression of insulin-like growth factor-1 in human pulp tissue according to the root-development stage.

Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation and differentiation. It actively participates during odontogenesis and mineralization of dental hard tissues. The purpose of this study is to use a radioimmunoassay (RIA) to determine whether there are differences in the expression of IGF-1 in human pulp from teeth having incomplete or complete root development. Twenty-six pulp samples were obtained from freshly extracted human third molars, half of them from teeth with incomplete root development. All of the samples were processed and labeled using an IGF-1 RIA kit. Results showed IGF-1 presence in all human pulp samples with a statistically significant higher expression in pulps having complete root development. The present findings are consistent with the hypothesis that IGF-1 contributes toward forming and mineralizing dental tissues in teeth with incomplete root development as well as in pulp-repairing process in teeth with complete root development.

Quantification of lactate-dehydrogenase and cell viability in postmortem human dental pulp.

Understanding pulp repair and regeneration requires being familiar with this tissue's behavior under extreme conditions, such as postmortem state where an abrupt interruption of tissue blood supply occurs. The purpose of this study was to quantify cell viability and the amount of lactate-dehydrogenase (LDH) expressed in human pulp tissue 6, 12, and 24 hours postmortem to establish how long dental pulp remains viable after death. Pulp samples were obtained from 14 unidentified corpses of people who had received lethal injuries in car accidents or from gunshot wounds; they had at least three caries- and restoration-free incisors. Half of each sample was used for determining cell viability at three different time intervals. The rest of each sample was used for quantifying LDH expression at the same time intervals. Another 14 pulp samples were obtained from live patients' healthy premolars where extraction was indicated for orthodontic reasons to assess normal LDH value in pulp tissue. The results showed cell viability decreasing from 89 to 68 to 41% measured 6, 12, and 24 hours postmortem, respectively. LDH expression in healthy pulps was 246 U/mg pulp weight. Expression increased after death from 249 U/mg at 6 hours to 337 U/mg at 12 hours. LDH expression decreased to 131 U/mg 24 hours postmortem. These findings are valuable in understanding dental pulp survival capability under extreme conditions that may have important clinical significance in terms of repair and regeneration.

The effect of cavity preparation on substance P expression in human dental pulp.

Substance P (SP) plays an important role during neurogenic inflammation of dental pulp. The purpose of this study was to use a radioimmunoassay for determining the effect of cavity preparation on SP expression in healthy human dental pulp. Ten pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Deep cavity preparation (<1 mm remaining dentine thickness) was performed before extraction in five of these bicuspids. All samples were processed and I-SP labeled; SP was quantified by competition assay. The results revealed SP expression in all human pulp samples. Mann-Whitney's U test revealed statistically significant higher expression in pulp from teeth where cavity preparation had been performed compared to control values (p < 0.05). These findings suggest that SP is released during common dental procedures (such as cavity preparation) and its expression may have an important clinical significance in terms of experiencing inflammation and pain.

Expression of insulin-like growth factor-1 receptor in human pulp tissue.

Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation and differentiation. The purpose of this study was to use a radioreceptor assay to evaluate whether IGF-1 receptors are present in human pulp and to determine whether differences in its expression are found in the pulp tissue of teeth having incomplete or complete root development. Twenty pulps were obtained from freshly extracted human third molars; they were then processed and labeled with I-IGF-1. The results showed IGF-1 receptor expression in all human pulp samples. t test revealed statistically significant higher expression in the pulps from teeth having incomplete root development (P <0.005). Given the functions of this growth factor system in other tissues, the present findings are consistent with the hypothesis that IGF-1 contributes toward forming and mineralizing dental tissues as well as in pulp-repairing processes.