Effect of non-steroidal anti-inflammatory drugs on pulpal and periapical inflammation induced by lipopolysaccharide.

OBJECTIVES: The objective of this study was to evaluate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) in controlling pulpal and periapical inflammation in vivo as a potential coadjutant systemic therapy for pulpitis. MATERIALS AND METHODS: A suspension containing E. coli lipopolysaccharide (LPS; 1.0 µg/µL) was inoculated into the pulp chamber of the first molars of C57BL/6 mice (n = 72), and the animals were treated daily with indomethacin or celecoxib throughout the experimental periods. After 7, 14, 21, and 28 days, the tissues were removed for histopathological, histoenzymology, histometric, and immunohistochemical evaluation. RESULTS: Inoculation of LPS into the pulp chamber induced the synthesis of the enzyme cyclooxygenase-2 (COX-2) in dental pulp and periapical region. Indomethacin and celecoxib treatment changed the profile of inflammatory cells recruited to dental pulp and to the periapex, which was characterized by a higher mononuclear cell infiltrate, compared to LPS inoculation alone which recruited a higher amount of polymorphonuclear neutrophils. Administration of indomethacin for 28 days resulted in the development of apical periodontitis and increased osteoclast recruitment, unlike celecoxib. CONCLUSIONS: NSAIDs indomethacin and celecoxib changed the recruitment of inflammatory cells to a mononuclear profile upon inoculation of LPS into the pup chamber, but indomethacin enhanced periapical bone loss whereas celecoxib did not. CLINICAL RELEVANCE: Celecoxib, a selective COX-2 inhibitor, can change the profile of inflammatory cells recruited to the dental pulp challenged with LPS and might a be potential systemic coadjutant for treatment of pulpitis.

Melatonin antagonizes lipopolysaccharide-induced pulpal fibroblast responses.

BACKGROUND: Pulpal inflammation is known to be mediated by multiple signaling pathways. However, whether melatonin plays regulatory roles in pulpal inflammation remains unclear. This study aimed at elucidating an in situ expression of melatonin and its receptors in human pulpal tissues, and the contribution of melatonin on the antagonism of lipopolysaccharide (LPS)-infected pulpal fibroblasts. METHODS: Melatonin expression in pulpal tissues harvested from healthy teeth was investigated by immunohistochemical staining. Its receptors, melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2), were also immunostained in pulpal tissues isolated from healthy teeth and inflamed teeth diagnosed with irreversible pulpitis. Morphometric analysis was subsequently performed. After LPS infection of cultured pulpal fibroblasts, cyclo-oxygenase (COX) and interleukin-1 ß (IL-1 ß) transcripts were examined by using reverse transcription-polymerase chain reaction (RT-PCR). Analysis of mRNA expression was performed to investigate an antagonism of LPS stimulation by melatonin via COX and IL-1 ß induction. Mann-Whitney U test and One-way ANOVA were used for statistical analysis to determine a significance level. RESULTS: Melatonin was expressed in healthy pulpal tissue within the odontoblastic zone, cell-rich zone, and in the pulpal connective tissue. Furthermore, in health, strong MT1 and MT2 expression was distributed similarly in all 3 pulpal zones. In contrast, during disease, expression of MT2 was reduced in inflamed pulpal tissues (P-value< 0.001), but not MT1 (P-value = 0.559). Co-culturing of melatonin with LPS resulted in the reduction of COX-2 and IL-1 ß expression in primary pulpal fibroblasts, indicating that melatonin may play an antagonistic role to LPS infection in pulpal fibroblasts. CONCLUSIONS: Human dental pulp abundantly expressed melatonin and its receptors MT1 and MT2 in the odontoblastic layers and pulpal connective tissue layers. Melatonin exerted antagonistic activity against LPS-mediated COX-2 and IL-1 ß induction in pulpal fibroblasts, suggesting its therapeutic potential for pulpal inflammation and a possible role of pulpal melatonin in an immunomodulation via functional melatonin receptors expressed in dental pulp.

Expression of Selected Inflammatory Mediators with Different Clinical Characteristics of Pulpal Inflammation.

INTRODUCTION: Accurately diagnosing the state of dental pulp is crucial when addressing tooth pain to determine the best treatment approach. This study aimed to investigate the concentration of inflammatory mediators in the dental pulp of mature teeth that have been exposed via caries but show no signs of apical periodontitis. METHODS: Samples of pulpal blood from adults with mature teeth responsive to pulp testing and have carious pulp exposures were obtained. These samples were analyzed for 12 inflammatory cytokines and other inflammatory proteins using the Luminex assay platform. Clinical factors were correlated with cytokine levels, and statistical analysis was performed to evaluate the impact of these factors on cytokine expression. RESULTS: Of the 36 patients that were included, 44.44% took pain medications, 33.33% had prolonged pulpal bleeding, 41.67% felt spontaneous pain, and 72.22% were diagnosed with symptomatic irreversible pulpitis. Significant correlations existed between presenting pain scores and levels of interleukin (IL)-1α, IL-6, and IL-8 (P < .05). Factors like analgesic medication intake, pain to percussion, pain to thermal testing, spontaneous pain, and nocturnal pain were significantly associated with higher levels of specific inflammatory proteins. No significant associations were observed with pain to palpation, bleeding time, or pulpal diagnosis. CONCLUSIONS: Inflammatory proteins, including cytokine levels may play a critical role in characterizing pulpal inflammation. Future studies should investigate the role of these potential biomarkers in determining the diagnosis of pulpitis and the prognosis of vital pulp therapy.

Design and evaluation of an MMP-9-responsive hydrogel for vital pulp therapy.

OBJECTIVE: To design and evaluate a matrix metalloproteinase 9 (MMP-9)-responsive hydrogel for vital pulp therapy. METHODS: A peptide linker with optimized sensitivity toward MMP-9 was crosslinked with 4-arm poly (ethylene glycol)-norbornene (PEG-NB) by thiol-norbornene photo-polymerization. This resulted in the formation of a hydrogel network in which the peptide IDR-1002 was incorporated. Hydrogel characterization and gelation kinetics were examined with Fourier-transform infrared spectroscopy, scanning electron microscopy, rheological testing, and swelling evaluation. Hydrogel degradation was examined through multiple exposure to pre-activated MMP-9, to simulate flare-ups of dental pulp inflammation. The IDR-1002 released from degraded hydrogels was measured with high-performance liquid chromatography. Effect of IDR-1002 released from hydrogels on one-week-old multispecies oral biofilms was evaluated using confocal laser scanning microscopy. RESULTS: MMP-9-responsive, injectable, and photo-crosslinkable hydrogels were successfully synthesized. When hydrogel degradation and release of IDR-1002 were examined with exposure to pre-activated MMP-9, IDR-1002 release was significantly correlated with elevated levels of MMP-9 (p < 0.05). The effectiveness of IDR-1002 in killing bacteria in multispecies oral biofilms was significantly enhanced when the hydrogels were immersed in 10 nM or 20 nM pre-activated MMP-9, compared to immersion in phosphate-buffered saline (p < 0.05). CONCLUSIONS: The MMP-9-responsive hydrogel is a promising candidate for on-demand delivery of bioactive agent in vital pulp therapy. CLINICAL SIGNIFICANCE: MMP-9 is one of the most important diagnostic and prognostic biomarkers for pulpitis. An MMP-9-responsive hydrogel has potential to be used as an in-situ on-demand release system for the diagnosis and treatment of dental pulp inflammation.

Evaluation of Pulp Repair after BiodentineTM Full Pulpotomy in a Rat Molar Model of Pulpitis.

Dental pulp is a dynamic tissue able to heal after injury under moderate inflammatory conditions. Our study aimed to evaluate pulp repair under inflammatory conditions in rats. For this purpose, we developed a rat model of controlled pulpitis followed by pulpotomy with a tricalcium silicate-based cement. Fifty-four cavities were prepared on the occlusal face of the maxillary upper first molar of 27 eight-week-old male rats. E. coli lipopolysaccharides at 10 mg/mL or phosphate-buffered saline PBS was injected after pulp injury. Non-inflamed molars were used as controls. Levels of inflammation-related molecules were measured 6 and 24 h after induction by enzyme-linked immunosorbent assay of coronal pulp samples. Pulp capping and coronal obturation after pulpotomy were performed with tricalcium silicate-based cement. Four and fifteen days after pulpotomy, histological and immunohistochemical analysis was performed to assess pulp inflammation and repair processes. Our results showed significantly higher levels of innate inflammatory proteins (IL-1ß, IL-6, TNF-α and CXCL-1) compared with those in controls. Moderate residual inflammation near the capping material was demonstrated by histology and immunohistochemistry, with the presence of few CD68-positive cells. We showed that, in this model of controlled pulpitis, pulpotomy with BiodentineTM allowed the synthesis at the injury site of a mineralized bridge formed from mineralized tissue secreted by cells displaying odontoblastic characteristics. Analysis of these data suggests overall that, with the limitations inherent to findings in animal models, pulpotomy with a silicate-based cement is a good treatment for controlling inflammation and enhancing repair in cases of controlled pulpitis.

Modeling Hypoxia Induced Factors to Treat Pulpal Inflammation and Drive Regeneration.

INTRODUCTION: The ability to resolve pulpal inflammation to achieve predictable regeneration of the dentin-pulp complex has remained elusive and presents a challenge for clinicians and researchers. Although the dentin-pulp complex can react naturally to injury by forming a bridge of reparative dentin that protects the pulp from further damage, this process is significantly impaired if inflammation persists. Because the secretion of inflammatory cytokines by injured pulpal cells causes significant pain and discomfort to patients, it is critical to resolve pulpal inflammation in a timely manner so as to create a microenvironment conducive for pulpal healing and reparative dentin formation. The emergent field of regenerative endodontics has encouraged the development and application of biologically driven therapies that take advantage of the intrinsic healing capacities of host cells within dental pulp and the periapical complex. METHODS: These studies were designed to test the hypothesis that exposure to hypoxic conditions can modulate the production of inflammatory cytokines/factors by mesenchymal cells in vitro. A multi-domain peptide hydrogel system that is highly conducive for the growth and differentiation of tooth-derived stem cells was used for these studies. Stem cells from human exfoliated deciduous teeth (SHEDs) were first cultured within 3-dimensional hydrogel constructs and then challenged with hypoxic stresses via addition of H2O2. RESULTS: MDP constructs were successfully generated, challenged with H2O2, decellularized and lyophilized, forming a potential biomaterial containing hypoxia induced repair molecules. The ability of cell-derived factors to convert the phenotype of lipopolysaccharide-primed macrophages from a proinflammatory to a pro-resolving state was examined in the presence of the lyophilized SHED cell constructs. CONCLUSIONS: Our data suggest that hypoxia induced SHED cell products can be captured within the hydrogel system and may be useful in the resolution of pulpal inflammation to create a favorable microenvironment for regeneration of the dentin-pulp complex.

Pulpal responses after direct pulp capping with two calcium-silicate cements in a rat model.

Bioactivity of Bio-MA, a calcium chloride accerelator-containing calcium-silicate cement, as a pulp capping material was evaluated on mechanically exposed rat molar pulp. Sixty maxillary first molars from Wistar rats were mechanically exposed and assigned to two capping materials: Bio-MA or white mineral trioxide aggregate (WMTA), and three periods: 1, 7, or 30 days. Nine molars were exposed and covered with polytetrafluoroethylene tape, as positive controls. From histological examination, inflammatory cell infiltration and reparative dentin formation were evaluated using grading scores. No significant difference in pulpal responses between the two materials was observed at any period (p>0.05). At 1 day, all experimental groups showed localized mild inflammation. At 7 days, dentin bridge was partially observed at exposure sites with few inflammatory cells. At 30 days, pulp appeared normal with complete tubular dentin bridges. Bio-MA with accerelator was biocompatible similar to WMTA and could be used as a pulp-capping material.

Effects of the exposure site on histological pulpal responses after direct capping with 2 calcium-silicate based cements in a rat model.

OBJECTIVES: Direct pulp capping is a treatment for mechanically exposed pulp in which a biocompatible capping material is used to preserve pulpal vitality. Biocompatibility tests in animal studies have used a variety of experimental protocols, particularly with regard to the exposure site. In this study, pulp exposure on the occlusal and mesial surfaces of molar teeth was investigated in a rat model. MATERIALS AND METHODS: A total of 58 maxillary first molars of Wistar rats were used. Forty molars were mechanically exposed and randomly assigned according to 3 factors: 1) the exposure site (occlusal or mesial), 2) the pulp-capping material (ProRoot White MTA or Bio-MA), and 3) 2 follow-up periods (1 day or 7 days) (n = 5 each). The pulp of 6 intact molars served as negative controls. The pulp of 12 molars was exposed without a capping material (n = 3 per exposure site for each period) and served as positive controls. Inflammatory cell infiltration and reparative dentin formation were histologically evaluated at 1 and 7 days using grading scores. RESULTS: At 1 day, localized mild inflammation was detected in most teeth in all experimental groups. At 7 days, continuous/discontinuous calcified bridges were formed at exposure sites with no or few inflammatory cells. No significant differences in pulpal response according to the exposure site or calcium-silicate cement were observed. CONCLUSIONS: The location of the exposure site had no effect on rat pulpal healing. However, mesial exposures could be performed easily, with more consistent results. The pulpal responses were not significantly different between the 2 capping materials.

Effect of Topical Epigallocatechin-Gallate on Lipopolysaccharide-induced Pulpal Inflammation in Rat Models.

Introduction: Pulpal inflammation can be marked by an increase in tumor necrosis factor-α (TNF- α), malondialdehyde (MDA) and calcitonin gene-related peptide (CGRP) level. Epigallocatechin-3-gallate (EGCG) demonstrates the ability to reduce cytokine expression, influence immune receptors, reduce inflammation, neutralize reactive oxygen species (ROS) and to inhibit pain conduction. The present research aimed to determine the anti-inflammatory, antioxidant and pain conduction inhibition of topical EGCG hydrogels in Lipopolysaccharide (LPS)-induced pulpal inflammation in rats. Methods and Materials: A total of 28 male Wistar rats were divided equally into four groups. The negative control group (N) received no treatment, while the positive control group (C) and the other two treatment groups (T1, T2) were induced with LPS for 6 h, followed by the application of topical polyethylene glycol (PEG) hydrogels for C group, 25 ppm EGCG hydrogels for T1 group and 75 ppm EGCG hydrogels for T2 group, before being filled with glass ionomer cement (GIC). After 24 h, PEG and EGCG were reapplied and refilled with GIC for 24 h. The pulp tissue samples were examined by means of immunohistochemistry (IHC) to identify TNF-α, MDA and CGRP expression. All the data obtained was analyzed with one-way analyses of variance (ANOVA) test. Results: The T1 and T2 groups showed a significant decrease in TNF-α and CGRP expression compared to the control group, but there was no significant decrease in MDA in either group (P<0.05). Conclusion: Based on the results of this study, topical application of 75 ppm EGCG hydrogels to the tooth cavities with six hours of pulpal inflammation has the optimal result in reducing the expression of TNF-α and CGRP, but not of MDA.

Effects of TGF-ß1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

Transforming growth factor-ß1 (TGF-ß1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-ß1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-ß1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-ß1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-ß1 slightly inhibited cell growth that was reversed by SB431542. TGF-ß1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-ß1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-ß1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-ß1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-ß1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-ß1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-ß1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Pulpal Tissue Inflammatory Reactions after Experimental Pulpal Exposure in Mice.

INTRODUCTION: The purpose of this study was to establish a stable experimental mice pulpal inflammatory model and to evaluate inflammatory reactions of pulpal tissue after pulpal exposure. METHODS: Pulpal inflammation was induced in 80 C57BL/6 mice by occlusal exposure of the pulp of the maxillary first molar. The mice were sacrificed randomly at 0, 1, 6, 12, 24, 48, and 72 hours after pulpal exposure. Mice without pulpal exposure served as controls. Maxillary teeth were obtained and prepared for histologic analyses and real-time polymerase chain reaction analyses. RESULTS: As the duration of pulpal exposure increases, the inflammatory reaction is exacerbated. Within 6 to 12 hours after pulpal exposure, pulp tissues experienced red blood cell extravasation to the destruction of the odontoblast layer. After 24 hours, necrosis was observed in the pulpal tissue; until 72 hours, necrosis spread to the whole coronal pulpal tissue, and a large number of inflammatory cells were found in the radicular pulpal tissue. The results of histomorphologic scores have the same trend; samples from the 72-hour group possessed the highest score followed by samples from other groups (P < .01). The expression levels of inflammatory cytokines increased over the 72 hours, and there was a high rate of inflammatory cytokine expression at 6 and 12 hours after pulpal exposure. CONCLUSIONS: Our study represents a stable mice model for studying pulpal inflammation in vivo. Mouse pupal inflammation progresses rapidly, with dramatic changes evident in just a few hours.

Reabsorción dentinaria interna coronaria: informe de un caso clínico / Coronary internal dentine resorption: report of a clinical case

Objetivo: La reabsorción dentinaria interna es un pro- ceso causado por la actividad odontoclástica asociada princi- palmente a la inflamación pulpar crónica y/o traumatismos, y se caracteriza por la pérdida progresiva de tejido dentinario y la posible invasión al cemento. El presente informe describe el diagnóstico y tratamiento de un molar inferior que presentó un cuadro sintomático de reabsorción dentinaria interna. Caso clínico: Un paciente de 38 años fue derivado a la consulta por presentar una zona de reabsorción interna en un segundo molar inferior. Durante el examen clínico y ra- diográfico se tomó una radiografía preoperatoria periapical con radiovisiógrafo en la que se observó la presencia de un área compatible con el diagnóstico de reabsorción dentinaria interna, el que fue posteriormente confirmado por medio de una tomografía computada de haz cónico. La imagen de la lesión se presentó como una zona radiolúcida deformante de bordes nítidos, localizada a nivel de la cámara pulpar. El tra- tamiento consistió en la extirpación de la pulpa coronaria y de la instrumentación, desinfección y obturación de los conduc- tos radiculares y la cavidad de acceso. En el control clínico y radiográfico realizado luego de 3 años se observó que el paciente estaba asintomático y las estructuras perirradiculares se encontraban dentro de los límites normales. La observación histológica del material removido de la cámara pulpar reveló la presencia de un tejido granulomatoso con numerosos vasos sanguíneos y escasos focos micro hemorrágicos. Hasta el momento, el tratamiento endodóntico es el pro- cedimiento indicado para el tratamiento de la reabsorción dentinaria interna. Se destaca la importancia de la tomografía computada de haz cónico para el diagnóstico y tratamiento temprano de las reabsorciones dentinarias internas a efectos de contar con un pronóstico favorable (AU)

Aim: Internal dentine resorption is aprocess caused by odontoclastic activity, mainly associated with chronic pulpal inflammation and/or trauma, and it'scharacterized by a pro- gressive loss of dentine tissue and the possible invasion of the cementum. This report describes the diagnosis and treatment of a lower molar that presented a symptomatic case of inter- nal dentine resorption. Clinical case: A 38-years old patient was referred to the office because of presenting an area of internal resorption in a lower second molar. During clinical and radiographic exam- ination, a periapical preoperative radiograph with radiovisio- graph was taken, in which the presence of an area compatible with the diagnosis of internal dentine resorption was observed, which was later confirmed by a cone-beam computed tomog- raphy.The image of the lesion was presented as a deforming radiolucent area with sharp edges, located at pulp chamber level. Treatment consisted of the removal of the coronary pulp and the instrumentation, disinfection and filling of the root ca- nals and the access cavity. In the clinical and radiographic control carried out 3 years after procedure, it was observed that the patient was asymptomatic and the periradicular struc- tures were within normal limits. The histological observation of the removed material from the pulp chamber revealed the presence of a granulomatous tissue with numerous blood ves- sels and scarce micro hemorrhagic focus. Until now, the endodontic treatment is the indicated pro- cedure to treat internal dentine resorption. It is necessary to highlight the importance of the cone-beam computerized to- mography for the early diagnosis and treatment of internal dentine resorptions in order to have a favorable outlook (AU)

"Internal root resorption: An endodontic challenge": A case series.

Management of internal root resorption is a challenge to the endodontists. It may occur in cases with chronic pulpal inflammation, following caries or due to trauma in the form of an accidental blow. Most cases of internal root resorption are seen in anterior teeth, due to their susceptibility to trauma. However, it may be seen in posterior teeth, most likely because of carious involvement of the pulp. Early diagnosis, removal of the cause, proper treatment of the resorbed root is mandatory for successful treatment outcome. This paper is an attempt to summarize the knowledge on internal root resorption and present various cases, which were successfully managed with different treatment modalities.