[Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats].

Objective: To investigate the effect and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats (hereinafter referred to as burn serum). Methods: Experimental research methods were adopted. Thirty gender equally balanced Wistar rats aged 7 to 8 weeks were collected, 10 of which were used to prepare normal rat serum (hereinafter referred to as normal serum), and the other 20 were inflicted with full-thickness burn of 30% total body surface area to prepare burn serum. Primary cardiomyocytes were isolated and cultured from the apical tissue of 180 Wistar rats aged 1 to 3 days by either gender for follow-up experiments. Cells were divided into normal serum group and burn serum group treated with corresponding serum according to the random number table (the same grouping method below). Trypanosoma blue staining was performed at post treatment hour (PTH) 1, 3, 6, 9, and 12 to detect the cell survival rate. Cells were divided into burn serum alone group treated with burn serum for 6 h followed by routine culture of 30 min and 0.4 mmol/L glycine group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, 1.6 mmol/L glycine group, and 2.0 mmol/L glycine group treated with burn serum for 6 h followed by culture of 30 min with corresponding final molarity of glycine, i.e., at post intervention hour (PIH) 6.5, the cell survival rate was detected as before. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group, with the same intervention of 6.5 h as before, respectively. The content of adenosine monophosphate (AMP) and adenosine triphosphate (ATP) was detected by high performance liquid chromatography, and the AMP/ATP ratio was calculated. The protein expressions of phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1), and phosphorylated AMP-activated protein kinase (p-AMPK) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group intervened as before and 0.8 mmol/L glycine+25 ng/mL rapamycin group treated with burn serum followed by culture with two reagents. The expressions of heat shock protein 70 (HSP70), metallothionein (MT), and tubulin were detected by immunofluorescence method after 30 min of corresponding culture at PTH 1, 3, and 6, i.e., at PIH 1.5, 3.5, and 6.5, and the microtubule morphology was observed at PIH 6.5. The sample number at each time point was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)-t test, LSD test, and Bonferroni correction. Results: At PTH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (with t values of 4.96, 16.83, 35.51, 34.33, and 27.88, P<0.05). In burn serum group, the cell survival rate at PTH 3, 6, 9, or 12 was significantly lower than that at PTH 1 (P<0.05), the cell survival rate at PTH 6, 9, or 12 was significantly lower than that at PTH 3 (P<0.05), and the cell survival rate at PTH 6 was similar to that at PTH 9 (P>0.05) but significantly higher than that at PTH 12 (P<0.05). Treatment of 6 h was selected as the follow-up intervention time of burn serum. At PIH 6.5, compared with that in burn serum alone group, the cell survival rate in each glycine group was significantly increased (P<0.05). The cell survival rate in 0.8 mmol/L glycine group was the highest, and 0.8, 1.2, and 1.6 mmol/L were selected as subsequent glycine intervention concentrations. At PIH 6.5, the AMP/ATP ratio of cells in burn serum alone group was significantly higher than that in normal serum group, 1.2 mmol/L glycine group, or 1.6 mmol/L glycine group (P values all <0.05), and the AMP/ATP ratio of cells in 1.6 mmol/L glycine group was significantly lower than that in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group were 1.001±0.037, 0.368±0.020, 1.153±0.019, 1.128±0.062, 1.028±0.037, 0.96±0.07, 0.63±0.12, 1.17±0.13, 1.13±0.16, 1.11±0.11, and 0.98±0.06, 0.45±0.08, 1.13±0.05, 0.77±0.12, 0.51±0.13. Compared with those in burn serum alone group, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group and each glycine group were significantly increased (P<0.05), while the protein expressions of p-AMPK were significantly decreased (P<0.05). Compared with those in 0.8 mmol/L glycine group, the protein expression of p-4E-BP1 of cells in 1.2 mmol/L glycine group and the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05). Compared with those in 1.2 mmol/L glycine group, the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05), while the protein expression of p-AMPK was significantly increased (P<0.05). Compared with those in normal serum group, the expression of tubulin of cells in burn serum alone group was significantly decreased at PIH 1.5, 3.5, and 6.5 (P<0.05), while the expression of HSP70 of cells at PIH 1.5 and 3.5 and the expression of MT at PIH 3.5 and 6.5 were significantly increased (P<0.05). The expressions of HSP70 and MT of cells at PIH 1.5, 3.5, and 6.5 and the expression of tubulin at PIH 1.5 and 3.5 in burn serum alone group and 0.8 mmol/L glycine+25 ng/mL rapamycin group were significantly lower than those in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, compared with that in normal serum group, the cell microtubule structure in burn serum alone group was disordered; the cell boundary in 0.8 mmol/L glycine group was clearer than that in burn serum alone group, and the microtubule structure arranged neatly near the nucleus. Compared with that in 0.8 mmol/L glycine group, 0.8 mmol/L glycine+25 ng/mL rapamycin group had unclear cell boundaries and disordered microtubule structure. Conclusions: Burn serum can cause cardiomyocytes damage in rats. Glycine can significantly up-regulate mammalian target of rapamycin/p70 ribosomal protein S6 kinase/eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway through AMP-activated protein kinase, promote the synthesis of protective proteins HSP70, MT, and tubulin, stabilize the microtubule structure, and exert cardiomyocytes protection function.

Human closed and open apex premolar teeth express different toll-like receptor.

BACKGROUND: The innate immune activation which promotes inflammation responses in the dental pulp tissue leads to the progression of dentin caries. Accordingly, toll-like receptors (TLRs) are key molecules of the innate immune system that identify pathogen-associated molecular patterns (PAMPs) on microorganisms and may have a critical role in a dental injury. Therefore, this study aimed to investigate the expression of TLR2, TLR3, and TLR4 in the human dental pulp of opened and closed apex teeth. METHODS: Human dental pulps were derived from the healthy opened and closed apex premolar, in which extraction was indicated for orthodontic reasons. The extraction of RNA was performed and the gene expression determined by real-time polymerase chain reaction (RT-PCR). The result from real-time PCR was confirmed using western blot analysis. RESULTS: Real-time PCR data analysis showed that the expression TLR2 and TLR4 were significantly increased in closed apex premolar teeth compared to open apex teeth, whereas TLR3 expression was not significantly different in these two groups (p < .05). CONCLUSION: The results of the present study suggested increased expression of TLR2 and TLR4 by the maturation of the apex, which may be due to the presence of microorganisms in the normal or destructed dental pulp tissue. Thus, identifying the expression of TLRs molecules in dental pulp tissue helps to develop a deeper knowledge of the immune responses in the oral cavity.

Prostaglandin E2 Affects Interleukin 6 and Monocyte Chemoattractant Protein 1/CCL2 Production by Cultured Stem Cells of Apical Papilla.

INTRODUCTION: Root canal treatment of immature necrotic teeth is a major challenge in current endodontics. The effect of inflammatory mediators, such as prostaglandin, on the modulation of stem cells of the apical papilla (SCAP) is not completely understood. The aim of this study was to investigate the role of prostaglandin E2 (PGE2) on SCAP activation by Escherichia coli lipopolysaccharide (LPS) in vitro. METHODS: SCAP cultures were established and characterized. Increasing concentrations of lipopolysaccharide (0.1-10 µg/mL) were used to investigate cyclooxygenase-2 (COX-2/PTGS2) and PGE2 receptors (EP1-4) gene expression. Then, SCAP were treated with a COX-2 inhibitor (indomethacin) before treatment with different concentrations of LPS. The levels of the chemokine CCL2/monocyte chemoattractant protein 1 and interleukin (IL)-6 were detected in cell supernatants (24 hours) by enzyme-linked immunosorbent assay. Data analysis was performed using analysis of variance followed by the Tukey post test. RESULTS: The expression of COX-2 was up-regulated in the group treated with LPS at 1µg/mL compared with that in the control group. EP1-4 were detected in all experimental conditions at similar levels. SCAP treated with indomethacin presented a down-regulation in the production of LPS-induced CCL2 and the secretion of IL-6. CONCLUSIONS: SCAP showed increased COX-2 (PTGS2) gene expression induced by LPS and a PGE2-dependent production of IL-6 and CCL2.

Association of clinical variables and polymorphisms in RANKL, RANK, and OPG genes with external apical root resorption.

OBJECTIVES: The aim of this study was to investigate the association of clinical variables and polymorphisms in the RANKL, RANK, and OPG genes with external apical root resorption (EARR). METHODS: The sample was composed of 338 unrelated patients of both sexes, average age 14.9 years (range 8-21) with Class II Division 1 malocclusion, orthodontically treated. Periapical radiographs of the maxillary central incisor with the longer root (reference tooth) were taken before treatment and 6 months after starting treatment. DNA was extracted from buccal epithelial cells with the use of 10 mol/L ammonium acetate and 1 mmol/L EDTA. The analysis of 42 polymorphisms in the RANKL, RANK, and OPG genes was performed by means of real-time polymerase chain reaction. Univariate and multivariate analyzes were performed to verify the association of clinical and genetic variables with EARR (P <0.05). RESULTS: The initial root length and patient age were associated with EARR. Considering the study of polymorphisms of RANKL, no significant association was found of genetic polymorphisms with EARR. For RANK polymorphisms, only rs12455775 was associated with EARR. Regarding OPG polymorphisms, an association of rs3102724, rs2875845, rs1032128, and rs3102728 with EARR was found. After multivariate analysis, the initial root length, rapid maxillary expansion, and rs3102724 of the OPG gene were associated with EARR. CONCLUSIONS: Longer roots of upper central incisors and rapid maxillary expansion, as well as allele A of the rs3102724 polymorphism of the OPG gene, were associated with EARR in the study population.

Is Hypoxia Related to External Cervical Resorption? A Case Report.

Despite the fact that external cervical resorption (ECR) is a well-known and rather frequently met condition, the driving force of this phenomenon still remains unclear. Recently, hypoxia has been linked to ECR. Thus, the aim of this work was to investigate the existence of hypoxia in ECR and hypothesize on its role at the time of extraction. This work is a case study of a tooth with ECR. ECR diagnosis was based on clinical and radiographic examination with cone-beam computed tomographic imaging. The extracted tooth was further analyzed by using nanofocus computed tomographic imaging and immunohistology. To investigate the 3-dimensional extent and pattern of ECR, in vivo cone-beam computed tomographic imaging and ex vivo nanofocus computed tomographic imaging were used. Different histologic stains were used to investigate the presence of a hypoxic environment and to gain a better insight into the involved cells, neuronal structures, and remodeling process during ECR. A higher distribution of hypoxia-inducible factor 1a-positive cells was found in the apical part of the resorption area when compared with the coronal area of the resorption. In addition, a similar distribution of hypoxia-inducible factor 1a-positive odontoblasts was observed in the pulp. Three-dimensional analysis of the calcification of the pulp revealed the formation of pulp stones in areas with higher hypoxia. Histology showed that remodeling during ECR can occur according to a layered pattern. This investigation confirms the presence of hypoxia in ECR and shows that there is a gradient of hypoxia within the ECR lesion and surrounding tooth structure. The hypoxic environment within the pulp is also indicated by the formation of pulp stones.

Expression of Toll-like receptors 2 and 4 and its association with matrix metalloproteinases in symptomatic and asymptomatic apical periodontitis.

To determine Toll-like receptors (TLR)2 and TLR4 expression levels and associate them with matrix metalloproteinases (MMPs) in asymptomatic apical periodontitis (AAP), symptomatic apical periodontitis (SAP), and healthy controls. Apical tissue/lesion samples were obtained from chronic AAP (n = 35) and SAP (n = 29), and healthy periodontal ligament (HPL, n = 10) with indication of tooth extraction, respectively. mRNA expression levels of TLR2, TLR4, MMP-1, MMP-2, MMP-8, and MMP-13 were determined by real-time reverse-transcription polymerase chain reaction. The data were analyzed with Kruskal-Wallis and Dunn's pot hoc test (p < 0.05). The correlation coefficient was obtained using the Spearman correlation (p < 0.05). TLR2, MMP-1, MMP-2, and MMP-13 mRNA levels were the highest in SAP followed by AAP and controls (p < 0.05). TLR4 and MMP-8 were over expressed in AAP and SAP compared to HPL (p < 0.05). TLR2 positively correlated with TLR4, MMP-1, MMP-8, and MMP-13 in SAP (p < 0.05). TLR2 and TLR4 are overexpressed in apical lesions versus healthy periodontal ligament and correlate with collagenolytic MMPs. Particularly, TLR2 is overexpressed in SAP in association with MMP-1, MMP-8, and MMP-13. Our results suggest that the activation of TLR2 along with MMP overexpression might contribute to SAP clinical presentation and progression. TLRs, MMPs, and their interaction can explain the clinical presentations and evolution of apical periodontitis and might represent key targets for new diagnostic and treatment approaches.

Cell migration capability of vascular endothelial growth factor into the root apex of a root canal model in vivo.

Once a tooth develops deep caries and the dental pulp tissue is irreversibly infected, the infected dental pulp tissue should be removed, and filling material should be placed in the root canal. Endodontically treated teeth are prone to root fracture or periapical periodontitis; however, dental pulp tissue has the potential to prevent root fracture or periapical periodontitis. Therefore, dental pulp regeneration after pulpectomy may help prolong tooth life. In this study, a new method of dental pulp regeneration was developed. Vascular endothelial growth factor-adsorbed collagen gel was injected into the root canal of a prepared root canal model, placed into the dorsum of a rat, and cultured for 3 weeks. After retrieving the implant, histological analysis was performed. It was found that rat somatic cells were recruited into the root apex of the transplanted root canal model. These findings suggest a new potential technique for engineering dental pulp tissue.

Evaluation of Apically Extruded Debris Using Positive and Negative Pressure Irrigation Systems in Association with Different Irrigants.

This study evaluated the amount of apically extruded debris after chemo-mechanical preparation (CMP) using positive and negative pressure irrigation systems [Conventional irrigation (CI) and EndoVac (EV)] in association with different irrigants [6% Sodium Hypochlorite (NaOCl), 2% Chlorhexidine gel + saline solution (CHXg + SS), 2% Chlorhexidine solution (CHXs) or Saline solution (SS)]. Eighty mandibular premolars with single root canals were selected and randomly assigned into 8 groups (n = 10) according to the irrigation system and the irrigant used during CMP: G1 (EV + NaOCl), G2 (EV + CHXg + SS), G3 (EV + CHXs), G4 (EV + SS), G5 (CI + NaOCl), G6 (CI + CHXg + SS), G7 (CI + CHXs) and G8 (CI + SS). Reciproc® R25 files (25/.08) were used during the CMP and the extruded debris from each tooth was collected in pre-weighted Eppendorf tubes and dried. The average weight of debris was assessed using a microbalance, and the data were statistically analyzed using ANOVA and the post hoc Tukey's test (a = 0.05). All groups were associated with debris extrusion. EV was the irrigation system with less extruded debris (p < 0.05). No differences were observed regarding the irrigant when EV was used. When CI was used, CHXg + SS were associated with lower debris extrusion (p < 0.05). It was concluded that no irrigation protocol succeeded in preventing debris extrusion. EV resulted in lower levels of debris extrusion than CI. The use of CHXg + SS resulted in lower debris extrusion.

Evaluation of apically extruded debris using positive and negative pressure irrigation systems in association with different irrigants

Abstract This study evaluated the amount of apically extruded debris after chemo-mechanical preparation (CMP) using positive and negative pressure irrigation systems [Conventional irrigation (CI) and EndoVac (EV)] in association with different irrigants [6% Sodium Hypochlorite (NaOCl), 2% Chlorhexidine gel + saline solution (CHXg + SS), 2% Chlorhexidine solution (CHXs) or Saline solution (SS)]. Eighty mandibular premolars with single root canals were selected and randomly assigned into 8 groups (n = 10) according to the irrigation system and the irrigant used during CMP: G1 (EV + NaOCl), G2 (EV + CHXg + SS), G3 (EV + CHXs), G4 (EV + SS), G5 (CI + NaOCl), G6 (CI + CHXg + SS), G7 (CI + CHXs) and G8 (CI + SS). Reciproc® R25 files (25/.08) were used during the CMP and the extruded debris from each tooth was collected in pre-weighted Eppendorf tubes and dried. The average weight of debris was assessed using a microbalance, and the data were statistically analyzed using ANOVA and the post hoc Tukey's test (a = 0.05). All groups were associated with debris extrusion. EV was the irrigation system with less extruded debris (p < 0.05). No differences were observed regarding the irrigant when EV was used. When CI was used, CHXg + SS were associated with lower debris extrusion (p < 0.05). It was concluded that no irrigation protocol succeeded in preventing debris extrusion. EV resulted in lower levels of debris extrusion than CI. The use of CHXg + SS resulted in lower debris extrusion.

Resumo Este estudo avaliou a quantidade de debris extruídos apicalmente após o preparo químico-mecânico (PQM) utilizando sistemas de irrigação com pressão positiva e negativa [irrigação convencional (IC) e EndoVac (EV)] em associação com diferentes irrigantes [hipoclorito de sódio 6% (NaOCl), clorexidina gel + solução salina (CLXg + SS), solução de clorexidina 2% (CLXs) ou solução salina (SS)]. Oitenta pré-molares inferiores com único canal radicular foram selecionados e aleatoriamente alocados em 8 grupos (n=10) de acordo com o sistema de irrigação e irrigante utilizado durante o PQM: G1 (EV + NaOCl), G2 (EV + CLXg + SS), G3 (EV + CLXs), G4 (EV + SS), G5 (IC + NaOCl), G6 (IC + CLXg + SS), G7 (IC + CLXs) e G8 (IC + SS). Limas Reciproc® R25 foram utilizadas durante o PQM e os debris extruídos de cada dente foi coletado em tubos pré-pesados e secos. O peso médio de debris foi avaliado por meio de microbalança, e os dados foram analisados estatisticamente utilizando ANOVA e teste de Tukey (a = 0.05). Todos os grupos foram associados com extrusão de debris. EV foi o sistema de irrigação com menos debris extruídos (p<0.05). Não foram observadas diferenças entre os irrigantes quando o EV foi utilizado. Quando foi utilizada IC, CLXg + SS foram associados a menor extrusão de debris (p<0.05). Concluiu-se que nenhum protocolo de irrigação conseguiu prevenir extrusão de debris. EV resultou em menores níveis de extrusão de debris que a IC. A utilização da CLXg + SS resultou em menor extrusão de debris.

Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis.

AIM: To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated. METHODOLOGY: Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant. RESULTS: All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found. CONCLUSION: Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis.

The Effects of Irrigants on the Survival of Human Stem Cells of the Apical Papilla, Including Endocyn.

INTRODUCTION: Endocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. METHODS: To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction. RESULTS: Endocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. CONCLUSION: Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn.

Expression of Mineralization Markers during Pulp Response to Biodentine and Mineral Trioxide Aggregate.

INTRODUCTION: The purpose of this study was to compare the cell viability of dental pulp cells treated with Biodentine (Septodont, Saint-Maur, France) and mineral trioxide aggregate (MTA) and the in vitro and in vivo expression of mineralization markers induced by the 2 materials. METHODS: Human dental pulp cells isolated from 6 permanent teeth were stimulated with Biodentine and MTA extracts. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and quantitative reverse-transcriptase polymerase chain reaction was used to determine the expression of mineralization markers. Specimens of teeth from dogs treated with Biodentine and MTA after pulpotomy were used to determine the presence of osteopontin and alkaline phosphatase by immunohistochemistry and runt-related transcription factor 2 by immunofluorescence. RESULTS: No significant differences in cell viability were found between MTA and Biodentine extracts and controls after 24 and 48 hours (P > .05). After 48 hours, osteopontin (SPP1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2) expression was higher in MTA and Biodentine than in controls (P < .05). Osteopontin staining was more intense and spread over a greater number of areas in Biodentine than in MTA samples (P < .0001). Alkaline phosphatase staining of a mineralized tissue bridge was significantly different between materials (P < .0001), but no difference in alkaline phosphatase staining of pulp tissue was found between MTA and Biodentine (P = .2). Also, no significant difference in the number of cells labeled for runt-related transcription factor 2 by immunofluorescence was observed between materials (P > .05). CONCLUSIONS: Biodentine stimulated similar markers as MTA, but staining was more intense and spread over a larger area of the pulp tissue.

Dentinal Tubule Penetration of Tricalcium Silicate Sealers.

INTRODUCTION: The treatments for which mineral trioxide aggregate (MTA)-based materials can be used in dentistry are expanding. Smaller particle size and easier handling properties have allowed the advent of tricalcium silicate sealers including EndoSequence BC Sealer (Brasseler USA, Savannah, GA), QuickSet2 (Avalon Biomed, Bradenton, FL), NeoMTA Plus (Avalon Biomed), and MTA Fillapex (Angelus, Londrina, Brazil). The objective of this study was to measure the tubule penetration with these sealers using continuous wave (CW) and single-cone (SC) obturation techniques. METHODS: Eighty single-rooted teeth were randomly divided into 8 groups of 10 and obturated with 1 of the previously mentioned sealers mixed with trace amounts of rhodamine using either the CW or SC technique. Teeth were sectioned at 1 mm and 5 mm from the apex and examined under a confocal laser microscope. The percentage of sealer penetration and the maximum sealer penetration were measured. RESULTS: The tricalcium silicate sealers penetrated tubules as deep as 2000 µm (2 mm). The percentage of sealer penetration was much higher 5 mm from the apex, with many specimens having 100% penetration for both SC and warm vertical techniques. MTA Fillapex, a resin-based sealer with less than 20% MTA particles, had significantly greater tubule penetration with a warm vertical technique versus the SC technique at the 1-mm level. CONCLUSIONS: Within the limitations of this study, the CW and SC techniques produced similar tubule penetration at both the 1-mm and the 5-mm level with the tricalcium silicate sealers BC Sealer, QuickSet2, and NeoMTA Plus.

CXC Chemokine Receptor 4 Is Expressed Paravascularly in Apical Papilla and Coordinates with Stromal Cell-derived Factor-1α during Transmigration of Stem Cells from Apical Papilla.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) at the apex may be attracted into the root canal space as a cell source for pulp-dentin regeneration. To test this possibility, we used in vitro transmigration models to investigate whether SCAPs can be chemoattracted by the delivery of the chemotactic cytokine stromal cell-derived factor-1α (SDF-1α). METHODS: We first examined the expression of CXC chemokine receptor 4 (CXCR4) for SDF-1α in the apical papilla and in cultured SCAPs using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and flow cytometric analyses. A standard Transwell migration assay and a 3-dimensional cell migration assay were used to analyze transmigration of SCAPs via the SDF-1α/CXCR4 axis. RESULTS: CXCR4 was expressed in the paravascular region of the apical papilla and detected in SCAP cultures. Most cultured SCAPs harbored intracellular CXCR4 (58%-99%, n = 4), whereas only a few cells had detectable CXCR4 on the cell surface (0.3%-2.34%, n = 4). Although SDF-1α had no significant effect on SCAP proliferation, it significantly promoted a higher number of migrated cells; this effect was abolished by anti-CXCR4 antibodies. Interestingly, cell surface CXCR4 on SCAPs was not detectable until after transmigration. The 3-dimensional migration assay revealed that SDF-1α significantly enhanced SCAP migration in the collagen gel. CONCLUSIONS: SCAPs can be chemoattracted via the SDF-1α/CXCR4 axis, suggesting that SDF-1α may be used clinically to induce CXCR4-expressing SCAPs in the apical papilla to transmigrate into the root canal space as an endogenous cell source for pulp regeneration.

Immunohistochemical localization of NALP3 inflammasome in experimental periapical lesions.

AIM: To explore the role of NALP3 inflammasome [NALP3, its effector molecule apoptosis associated speck-like protein (ASC), caspase-1, interleukin (IL)-1ß and IL-18] in the development of periapical lesions in rats. METHODOLOGY: Periapical lesions were developed within 21 days after mandibular first molar pulp exposure in Sprague-Dawley rats. The animals were randomly sacrificed at 0, 1, 3, 7, 10, 14 and 21 days after pulpal exposure. The bilateral mandibles were extracted for histological processing, then they were haematoxylin-eosin (HE) stained to examine inflammation infiltration in the apical region and immunohistochemically examined for the NALP3 inflammasome signalling pathway. Data were analysed by one-way analysis of variance and the Pearson(') s correlation and linear tendency test. RESULTS: NALP3 was detected in the cytoplasm of fibroblasts, monocytes, neutrophils, macrophages and vascular endothelial cells in the periapical region. From day 1 to day 21, the number of NALP3-positive cells ascended and was significantly correlated with the intensity of inflammatory infiltration (r = 0.776, P < 0.01). ASC, caspase-1, IL-1ß and IL-18 were all expressed in the inflammatory periapical tissues. The positive cell counts of IL-1ß and IL-18 were significantly correlated with that of NALP3, and r = 0.718, P < 0.01; r = 0.688, P < 0.01, respectively. CONCLUSIONS: NALP3 inflammasome is expressed in the inflammatory periapical tissues. This cytokine-signalling pathway may therefore be crucial in the regulatory control of inflammatory responses in periapical tissues and the development of periapical lesions.

Comparison of irrigant penetration up to working length and into simulated lateral canals using various irrigating techniques.

AIM: To evaluate the effect of an apical negative pressure system, a passive ultrasonic irrigation system and a combination of both apical negative pressure and passive ultrasonic irrigation on the penetration of the irrigating contrast solution (ICS) up to working length and into simulated lateral canals. METHODOLOGY: The root canals of 64 single-rooted teeth were instrumented using the ProTaper rotary system. In each sample, three simulated lateral canals were created at 2, 4 and 6 mm levels from the root apex using a 06-size C+ file (Dentsply Maillefer, Ballaigues, Switzerland). Samples were randomly assigned into 4 experimental groups (n = 16): group I - conventional needle irrigation, group II - passive ultrasonic irrigation, group III - apical negative irrigation system and group IV - combination of passive ultrasonic irrigation and apical negative pressure irrigation system. To examine irrigating solution penetration, Indian ink was mixed with 5.25% NaOCl and delivered into the root canals. Samples were then assessed by direct observation of the images taken using Canon EOS rebel T3. The depth of penetration of ICS up to the working length and into the simulated lateral canals was analysed using chi-squared tests. RESULTS: The combination (ANP and PUI) and ANP group had significantly deeper ICS penetration up to the working length (P < 0.001). The combination (ANP and PUI) and the PUI group exhibited significantly greater ICS penetration into lateral canals at the 6 mm level (P < 0.001). At the 4 and 2 mm levels, the combination of ANP and PUI had significantly greater ICS penetration into the lateral canals than the other groups (P < 0.001). CONCLUSIONS: The combination of ANP and PUI was the only group able to achieve irrigating contrast solution penetration both up to the working length and into lateral canals.

Localization of 14C-labeled 2% lidocaine hydrochloride after intraosseous anesthesia in the rabbit.

OBJECTIVE: The purpose of this study was to investigate the tissue distribution of lidocaine hydrochloride in mandibular bone marrow after intraosseous anesthesia (IOA) in rabbits. METHODS: We used macroautoradiography to examine the tissue distribution of a (14)C-labeled 2% lidocaine hydrochloride solution containing 1:80,000 epinephrine ((14)C-lidocaine). Under general anesthesia, (14)C-lidocaine was injected intraosseously or paraperiosteally. After IOA, animals were divided into three groups and observed at 1 (IOA-1), 5 (IOA-5), and 10 minutes (IOA-10) after injection. After infiltration anesthesia (IA), animals were observed at 1 minute after injection. RESULTS: The accumulation of (14)C-lidocaine was observed around the injection site in both the IA and the IOA groups. Paraperiosteally injected (14)C-lidocaine diffused to the surrounding tissues such as the lip, whereas IOA showed concentrated accumulation around the root apex throughout the experiment. The distribution area was significantly smaller in the IOA-1 group than in the IA group. The distribution area in the IOA-5 group was larger than those in the IOA-1 and IOA-10 groups. CONCLUSIONS: The accumulation of (14)C-lidocaine injected by IOA in rabbits was concentrated around the root apex. These results may explain the rapid onset time of IOA.

Immunolocalization and expression of Runx2 in tertiary dentinogenesis.

Runx2 is a new transcription factor that takes part in odontoblast differentiation. This study is aimed at investigating the immunolocalization and expression of Runx2 in the process of dental pulp injury and repair using immunohistochemical technique. In normal dental pulp, positive staining can hardly be detected. In experimental groups, strong positive staining was detected at the site of the impaired pulp after 1 day, while only weak Runx2 staining was detected 3 days after operation. Five days later, a large number of stellate cells in the root apex expressed Runx2, and after 7 days, followed by the reparative dentinogenesis, Runx2 expression vanished slowly, then totally disappeared. Taken together, the expression of Runx2 has temporal and spatial specificity during different phases in rat tertiary dentinogenesis.

Matrix metalloproteinase expression in teeth with apical periodontitis is differentially modulated by the modality of root canal treatment.

INTRODUCTION: The objective of this study was to investigate the expression of matrix metalloproteinases (MMPs) in apical periodontitis and during the periapical healing phase after root canal treatment. METHODS: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. RESULTS: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. CONCLUSIONS: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes.

Influence of tooth age and root section on root dentine dye penetration.

AIM: To investigate dye permeability of root dentine according to patients' age, root section and dye penetration time. METHODOLOGY: A total of 96 extracted human single-rooted teeth, assigned to four age groups (<30, 30-45, 45-60 and >60 years) were separated at the cemento-enamel junction and root canals were enlarged. The root surfaces were coated with cyanocrylate to prevent external dye penetration and centrifuged in distilled water to eliminate air. For dye penetration the root canals were filled with methylene blue 5%. After 1, 30 and 60 days eight roots per age group were cross-sectioned in 1 mm slices. Dye penetrated areas and the complete dentine areas were digitized and measured. Differences between groups were judged with anova and LSD, P < 0.05 or P < 0.01. RESULTS: The root section, the patients' age and the penetration time influenced significantly the penetrated areas (P < 0.05). After 1 and 30 days significant differences could be found only in the apical root sections between all age groups (P < 0.05). Dye penetration areas systematically decreased with increasing age and also from coronal to apical (P < 0.01). CONCLUSIONS: Age influenced dye penetration significantly. Dye penetration also depended on the location (coronal, middle and apical) within the root canal. These findings indicate that there may be a correlation between the tooth age and permeability of root dentine, which may influence the distribution and effectiveness of drugs used for root canal disinfection.