Simvastatin Induces Apoptosis but Attenuates Migration in SCAPs.

AIM: Simvastatin has emerged as having a promising role in controlling stem cell behaviours. This study aimed to evaluate the effects of simvastatin on the viability, growth, and migration of stem cells isolated from apical papillae (SCAPs) in vitro. METHODS: SCAPs were isolated and characterised. The viability and proliferation were assessed using live/dead and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, respectively. Cell migration was evaluated using scratch assays. Cell cycle progression and apoptosis were examined using flow cytometry analysis. RESULTS: Simvastatin at a concentration of 100 to 1000 nM did not exhibit cytotoxicity. Simvastatin reduced cell numbers at days 3 and 7. In addition, simvastatin markedly decreased colony formation in both colony number and cell density in a dose-dependent manner. An increase in apoptosis was observed at day 7. There was statistically significant increased in sub G0 population. An in vitro cell migration was attenuated in a dose-dependent manner. CONCLUSION: Simvastatin affects SCAPs' viability, proliferation, and cell migration. The reduction of cell viability at day 7 could be due to apoptotic induction.

Poor neck posture and longer working duration during root canal treatment correlated with increased neck discomfort in dentists with <5-years' experience in endodontics.

OBJECTIVE: This study investigated the effects of neck posture and working duration during each step of root canal treatment (i.e. opening the canal [OC], length determination, mechanical instrumentation, try main cone, and filling the root canal) on neck discomfort (ND) in dentists with <5-years' endodontic experience. METHODS: Twenty-four dentists performed a one-visit endodontic treatment of an upper molar in a phantom head model. A video was recorded to evaluate the dentistsá¾½ neck postures using the Modified-Dental Operator Posture Assessment Instrument (M-DOPAI) and treatment duration. The M-DOPAI divides the dentistsá¾½ neck postures into three categories: acceptable, compromised, or harmful posture. The participants rated their ND using Borgá¾½s CR-10 scale every 10 min. and at the end of each treatment step. The relationships between neck posture/treatment duration and Borgá¾½s CR-10 scores were examined using partial correlation. RESULTS: The number of compromised and harmful neck postures during the endodontic procedure (r = 0.43, P = .04) and treatment duration (r = 0.58 P = .005) significantly correlated with ND at the end of treatment. The number of compromised and harmful neck postures during the OC step (r = 0.75, P < .001) and the duration of the OC step (r = .70, P < .001) significantly correlated with ND at the end of the step. CONCLUSION: Poor neck postures and long working duration during endodontic treatment correlated with ND among inexperienced dentists. Neck pain interventions should focus on neck postures and work duration during root canal treatment, particularly in the OC step.

Up-regulation of p38 mitogen-activated protein kinase during pulp injury-induced glial cell/neuronal interaction in the rat thalamus.

INTRODUCTION: We have recently reported that the signal of pulp injury induces both neuronal and glial cell activation in the contralateral thalamus in rats, although the mechanisms of the glial cell/neuronal interaction remain unclear. This study was undertaken to test our hypothesis that p38 mitogen-activated protein kinase (MAPK) signaling pathways are involved in the pulp injury-induced glial cell/neuronal interaction in the thalamus. METHODS: A local anesthetic (lidocaine with epinephrine) or saline (control) was injected into the tissue surrounding the left mandibular first molar of Wistar rats. The tooth was then pulp-exposed, and the cavity was sealed with flowable composite. After 0 (normal pulp with local anesthetic or saline pretreatment), 24, and 72 hours, the contralateral side of thalamus was retrieved and subjected to immunohistochemistry for phospho-p38 MAPK and glial fibrillary acidic protein and real-time polymerase chain reaction analysis of p38-MAPK family (MAPK 13 and MAPK 14) mRNAs. RESULTS: The area immunopositive to phospho-p38 MAPK increased until 72 hours after pulp exposure in both local anesthetic-pretreated and saline-pretreated animals, but the rate of increase was lower in the local anesthetic-pretreated animals. The density of glial fibrillary acidic protein-expressing astrocytes showed a significant increase only in the saline-pretreated animals. Expression levels of MAPK 13 and MAPK 14 mRNAs increased at 24 hours and still higher at 72 hours in the saline-pretreated animals. Notably, MAPK 13 and MAPK 14 mRNA levels at 24 and 72 hours in the local anesthetic-pretreated animals showed significantly lower levels than those in the saline-pretreated animals. CONCLUSIONS: It was concluded that pulp injury-induced up-regulation of MAPK 13, MAPK 14, and phospho-p38 MAPK in the thalamus was suppressed by the local anesthetic pretreatment, suggesting the involvement of p38 MAPK signaling pathways in the glial cell-neuronal interaction induced by pulpal nociception.

A novel whole tooth-in-jaw-bone culture of rat molars: morphological, immunohistochemical, and laser capture microdissection analysis.

In conventional whole-tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth-in-jaw-bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2-positive macrophages were also analyzed for their Class II MHC, interleukin-6 (IL-6), and p53 mRNA expression levels by means of immune-laser capture microdissection (immune-LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline-perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium-perfused explants in contrast to the cultured control groups. The Class II MHC, IL-6, and p53 mRNA expression levels of ED2-positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium-perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth-in-jaw-bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp.

Gene expression analysis of resident macrophages in lipopolysaccharide-stimulated rat molar pulps.

INTRODUCTION: In normal dental pulp, a considerable number of resident macrophages are distributed. This study was designed to analyze the expression levels of genes associated with differentiation and function of resident macrophages in rat molar pulps stimulated with lipopolysaccharide (LPS). METHODS: Mandibular first molars of 7-week-old male Wistar rats were used. After transcardiac perfusion with a culture medium to preserve tissue integrity, pulpotomy and LPS application were carried out on the experimental teeth, and then dissected mandibles were subjected to whole-tooth culture for 3 days. Normal teeth and pulpotomized teeth without LPS served as controls. The specimens were then immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, a resident macrophage marker). Real-time polymerase chain reaction for Toll-like receptor 4 (TLR4), CD14, chemokine receptors (CCR2 and CX3CR1), and colony-stimulating factor-1 (CSF1) mRNAs was carried out after laser capture microdissection of ED1+ and ED2+ cells. RESULTS: LPS-treated pulps showed significant increases in (1) density of ED1+ and ED2+ cells beneath the amputation site and (2) expression levels of TLR4, CD14, CSF1, and CX3CR1 mRNAs, as compared with non-LPS-treated groups. CCR2 mRNA showed no significant difference between each group. CONCLUSIONS: LPS treatment of cultured rat molars caused the accumulation of resident macrophages and enhanced the expression of TLR4, CD14, CSF1, and CX3CR1 mRNAs in these cells. Up-regulation of these molecules might be involved in the differentiation and subsequent migration of resident macrophages of the pulp.

The role of N-methyl-D-aspartate receptor subunits in the rat thalamic mediodorsal nucleus during central sensitization.

We have identified tooth pulp-driven neurons (TPDNs) in the thalamic mediodorsal nucleus (MD) in rats and showed that the TPDNs' responsiveness in the MD is increased by chemical conditioning stimulation of allyl-isothiocyanate (mustard oil) to the molar tooth pulp. The aim of the present study was to address the role of N-methyl-d-aspartate receptors (NMDA receptors) in the sensitized central nervous system following the mustard oil application to the rat tooth pulp. Microinjection of MK-801, a noncompetitive NMDA receptor antagonist, to the thalamic MD nucleus reduced the TPDNs' responsiveness in the thalamic MD nucleus. Gene expression analysis showed that expression levels of NMDA receptor subunits NR2A and NR2D mRNAs in the thalamus were increased by the mustard oil application and that the increases were reduced by MK-801. When naloxone, an opioid receptor antagonist, was given systemically following the MK801 microinjection, the TPDNs' responsiveness was rekindled and expression levels of NR2D and NR2A mRNAs were increased. Moreover, lidocaine pretreatment abolished the mustard oil-induced upregulation of NR2D and NR2A mRNAs. These results suggest that, during central sensitization, interaction of NMDA receptors and endogeneous opioid-related inhibitory mechanisms plays critical role in the alteration of the TPDNs' responsiveness in the thalamic MD nucleus.

Increased gene expression of Toll-like receptors and antigen-presenting cell-related molecules in the onset of experimentally induced furcation lesions of endodontic origin in rat molars.

INTRODUCTION: Early immunopathogenic mechanisms behind pulp infection-induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection-induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. METHODS: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. RESULTS: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. CONCLUSIONS: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation.

Artificial dental pulp exposure injury up-regulates antigen-presenting cell-related molecules in rat central nervous system.

INTRODUCTION: Bacterial infection and resulting inflammation of the dental pulp might not only trigger neuroimmune interactions in this tissue but also sensitize the central nervous system (CNS) such as the thalamus via nociceptive neurons. Thus, immunopathologic changes in the rat thalamus that take place after pulp inflammation were investigated. METHODS: Pulp exposure was made in mandibular right first molars of 5-week-old Wistar rats. After 24 hours, the thalamus was retrieved and subjected to either immunohistochemistry for class II major histocompatibility complex (MHC) molecules and glial fibrillary acidic protein (GFAP) or mRNA expression analysis of antigen-presenting cell-related molecules and N-methyl-D-aspartate receptor 2D subunit (NR2D) by means of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: At 24 hours after pulp exposure, the density of class II MHC molecule-expressing and GFAP-expressing cells was increased in the contralateral thalamus. Gene expression analysis revealed the up-regulation of class II MHC molecules, CD80, CD83, CD86, and NR2D in the contralateral thalamus, as compared with the ipsilateral thalamus. CONCLUSIONS: These results suggest the signal of pulp inflammation induces neuronal activation in the CNS.

Laser capture microdissection in dentistry.

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.