Angiogenesis during coronal pulp regeneration using rat dental pulp cells: Neovascularization in rat molars in vivo and proangiogenic dental pulp cell-endothelial cell interactions in vitro.

Background/purpose: Angiogenesis is considered a crucial event for dental pulp regeneration. The purpose of this study was to demonstrate neovascularization during coronal pulp regeneration in rat molars using rat dental pulp cells (rDPCs) and to examine whether rDPC-endothelial cell interactions promote proangiogenic capacity in vitro. Materials and methods: Maxillary first molars of Wistar rats (n = 42) were pulpotomized and rDPCs isolated from incisors were implanted with a porous poly (l-lactic acid) (PLLA) scaffold and hydrogel (Matrigel). After 3, 7, and 14 days, coronal pulp tissues were examined histologically and by nestin and CD146 immunohistochemistry. rDPCs and rat dermal microvascular endothelial cells (rDMECs) were cocultured for 4 days and vascular endothelial growth factor (VEGF) synthesis and angiogenic factor gene expression were determined by enzyme-linked immunosorbent assays and real-time polymerase chain reaction, respectively. Effects of cocultured medium on tube formation by rDMECs were also evaluated. Results: Implantation of rDPC/PLLA/Matrigel induced coronal pulp regeneration with dentin bridge formation and arrangement of nestin-positive odontoblast-like cells at 14 days. PLLA/Matrigel without rDPCs did not induce pulp regeneration. CD146-positive blood vessels increased in density in the remaining pulp tissues at 3 and 7 days, and in the regenerated pulp tissue at 14 days. rDPC/DMEC coculture significantly promoted VEGF secretion and mRNA expression of nuclear factor-kappa B, angiogenic chemokine CXCL1, and chemokine receptor CXCR1. Cocultured medium significantly promoted tube formation. Conclusion: Coronal pulp regeneration with rDPC/PLLA/Matrigel was accompanied by neovascularization. rDPC-rDMEC interactions may promote angiogenic activity represented by proangiogenic factor upregulation and tube formation in vitro.

Pulp inflammation induces Kv1.1 K+ channel down-regulation in rat thalamus.

OBJECTIVES: Signals from inflamed tooth pulp activate thalamic neurons to evoke central sensitization. We aimed to gain insights into the mechanisms mediating the early phase of pulpal inflammation-induced thalamic neural and glial activation. MATERIALS AND METHODS: Pulpal inflammation was induced via the application of mustard oil (MO) to the upper first molar of Wistar rats with local anesthesia (LA) or saline injection. After 0.5, 1, 2, and 24 hr, contralateral thalami were subjected to microarrays, a real-time polymerase chain reaction and immunohistochemistry to identify differentially expressed genes and assess potassium voltage-gated channel subfamily A member 1 (Kv1.1)-expressing axons and glial fibrillary acidic protein (GFAP)-expressing astrocytes. RESULTS: The Kv1.1 gene (Kcna1) was down-regulated and the density of Kv1.1-expressing axons decreased in non-anesthetized rats, but not in anesthetized rats 1 hr after the MO treatment. The density of GFAP-expressing astrocytes increased in both groups until 24 hr after the MO treatment, with a greater increase being observed in the saline-injection group than in the LA group. CONCLUSIONS: MO induced the transient down-regulation of Kcna1, transiently reduced the density of Kv1.1-expressing axons, and increased astrocytes in thalami within 1 hr of pulpal application. These results suggest central sensitization represented by neuronal hyperexcitability and astrocyte activation.

Neural Regeneration/Remodeling in Engineered Coronal Pulp Tissue in the Rat Molar.

INTRODUCTION: This study aimed to examine the process of reinnervation during coronal pulp tissue regeneration in a rat model in which rat bone marrow mesenchymal stem cells were implanted in pulpotomized molars. METHODS: The maxillary first molars of Wistar rats were pulpotomized, and preformed biodegradable porous poly L-lactic acid scaffolds and hydrogel carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7, and 14 days, the implanted teeth were processed for histologic analysis; immunoperoxidase staining for protein gene product 9.5 (a general neuronal marker), calcitonin gene-related peptide (CGRP), or substance P (SP); and real-time polymerase chain reaction for nerve growth factor (NGF) and growth-associated protein 43 (GAP-43) messenger RNA (mRNA) expression. RESULTS: Histologic analysis of the implanted region revealed sparse cellular distribution at 3 days, pulplike tissue with a thin dentin bridge-like structure at 7 days, and dentin bridge-like mineralized tissue formation and resorption of most scaffolds at 14 days. Protein gene product 9.5 and CGRP-immunoreactive nerve fibers showed the lowest density at 3 days and significantly increased until 14 days when the CGRP-immunoreactive fibers reached normal levels. SP-immunoreactive nerve fibers showed the highest density at 7 days and decreased to normal levels at 14 days. NGF mRNA increased with time, whereas GAP-43 mRNA levels peaked at 3 days and subsequently dropped until 14 days. CONCLUSIONS: Regeneration/remodeling of SP-immunoreactive and CGRP-immunoreactive nerve fibers with increased mRNA expression of NGF and GAP-43 occurred in a rat model of coronal pulp tissue engineering with bone marrow mesenchymal stem cells.

Crosstalk between dental pulp stem cells and endothelial cells augments angiogenic factor expression.

OBJECTIVES: We aimed to investigate whether the mesenchymal stem cell-endothelial cell crosstalk enhances angiogenic factor expression via nuclear factor-kappa B (NF-κB)-dependent mechanisms. MATERIALS AND METHODS: Human dermal microvascular endothelial cells (HDMECs) and stem cells from human exfoliated deciduous teeth (SHEDs) were cocultured for 96 hr, in the presence of NF-κB decoy oligodeoxynucleotides (ODNs) or scramble (control). Vascular endothelial cell growth factor (VEGF) and phospho-NF-κB p65 were measured with enzyme-linked immunosorbent assay. Angiogenesis-related gene expression was analyzed with microarray analysis followed by real-time polymerase chain reaction. Tube formation assay was conducted in the presence of NF-κB decoy. RESULTS: The VEGF and phospho-NF-κB p65 levels were significantly higher in the coculture with NF-κB decoy scramble than in single culture and coculture with NF-κB decoy ODN. Microarray analysis of SHEDs and HDMECs with NF-κB decoy scramble showed higher expression of proangiogenic genes, Bcl-2, NF-κB1, VEGFA, CXCL8, and CXCR1, and lower expression of proapoptotic genes, Bax and Caspase 9, compared to cells with NF-κB decoy ODN. Real-time PCR results for Bcl-2 and CXCL8 showed a similar trend. Tube formation assay showed more tube development in the presence of NF-κB decoy scramble. CONCLUSION: The SHED-HDMEC crosstalk enhanced proangiogenic factor expression via NF-κB-dependent pathways.

In vivo fate of bone marrow mesenchymal stem cells implanted into rat pulpotomized molars.

In our previous work, we established an in vivo coronal pulp regeneration model in which biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells (BM-MSCs) were implanted in the coronal pulp chamber of pulpotomized rat maxillary first molars. In this study, we investigated the in vivo fate of LacZ-labeled BM-MSCs in our coronal pulp regeneration model. BM-MSCs were nucleofected with pVectOZ-LacZ plasmid encoding ß-galactosidase 1 day before implantation, and the LacZ-transfected BM-MSCs were implanted into the pulpotomized pulp chamber with biodegradable preformed scaffold-hydrogel constructs. Empty vector was used as a control. After 3 and 14 days, the molars were retrieved and subjected to ß-galactosidase staining. At 3 days, ß-galactosidase-expressing cells with a round profile were located mainly around the scaffold. At 14 days, when the pulp-like tissue had been generated, the majority of ß-galactosidase-expressing cells were detected under the newly formed dentin bridge-like structure, where nestin-expressing odontoblast-like cells were arranged. Immunoreactivity for dentin sialoprotein, a marker of mature odontoblasts, was strongly detected under the original dentin. No ß-galactosidase staining was observed in the control group. Thus, we demonstrated that BM-MSCs survived for 2 weeks after implantation and colonized within the site of potential cytodifferentiation. Our findings indicated that BM-MSCs could differentiate into cells involved in mineralized tissue formation in the functionally relevant region.

Inhibition of Nuclear Factor Kappa B Prevents the Development of Experimental Periapical Lesions.

INTRODUCTION: Nuclear factor kappa B (NF-κB) is an important transcriptional regulator of angiogenesis involving B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) signaling pathways. Thus, inhibition of NF-κB may suppress the development of periapical lesions via blockage of angiogenesis. Accordingly, we examined the effects of NF-κB decoy oligodeoxynucleotide (ODN) treatment on experimentally induced periapical lesions. METHODS: Periapical lesions were induced in the mandibular first molars of 5-week-old male Wistar rats by the application of lipopolysaccharide to the pulp. NF-κB decoy ODN or NF-κB decoy scramble (control) was injected intraperitoneally every 7 days, starting 1 day before pulp exposure. After 28 days, the samples were retrieved, and digital radiographs were taken for radiomorphometry. Samples were processed for (1) immunohistochemistry of CD31, Bcl-2, and Bax; (2) laser capture microdissection to analyze Bcl-2, Bax, chemokine (C-X-C motif) ligand 1 (CXCL1), CXC receptor 2 (CXCR2), and vascular endothelial cell growth factor receptor 2 (VEGFR2) messenger RNA (mRNA) expression in CD31+ endothelial cells; (3) enzyme-linked immunosorbent assay to determine NF-κB/p65 activity; and (4) Western blotting for vascular endothelial growth factor expression. RESULTS: NF-κB decoy ODN treatment significantly reduced lesion size, NF-κB/p65 activity, and the density of CD31+ endothelial cells in the lesion. NF-κB decoy ODNs also down-regulated CXCL1, CXCR2, and VEGFR2 mRNAs and up-regulated Bax mRNA in endothelial cells but did not affect Bcl2 mRNA in endothelial cells. Vascular endothelial growth factor protein expression in the lesions was significantly decreased. CONCLUSIONS: The inhibition of NF-κB activity by decoy ODN treatment suppressed the development of experimentally induced periapical lesions with a concomitant reduction in angiogenic responses in endothelial cells.

Dental Pulp Tissue Engineering Using Mesenchymal Stem Cells: a Review with a Protocol.

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from human and animal sources such as rats. Recently, an in vivo protocol for pulp tissue engineering using implantation of bone marrow MSCs into rat pulpotomized molars was established by our research group. This coronal pulp regeneration model showed almost complete regeneration/healing with dentin bridge formation when the cavity was sealed with mineral trioxide aggregate (MTA) to create a biocompatible seal of the pulp. This method is a powerful tool for elucidating the processes of dental pulp tissue regeneration following implantation of MSCs. In the present review, we discuss the literature in the field of dental pulp tissue engineering using MSCs including dental pulp stem cells and stem cells from exfoliated deciduous teeth. In addition, we present a brief step-by-step protocol of the coronal pulp regeneration model focusing on the implantation of rat bone marrow MSCs, biodegradable scaffolds, and hydrogels in pulpotomized rat molars. The protocol may lay the foundation for studies aiming at defining further histological and molecular mechanism of the rat pulp tissue engineering.

Implantation of Endothelial Cells with Mesenchymal Stem Cells Accelerates Dental Pulp Tissue Regeneration/Healing in Pulpotomized Rat Molars.

INTRODUCTION: This study aimed to examine whether the implantation of mesenchymal stem cells (MSCs) with endothelial cells (ECs) accelerates pulp tissue regeneration/healing and induces dentin bridge formation in a rat model of molar coronal pulp regeneration. METHODS: The maxillary first molars of Wistar rats were subjected to pulpotomy. Then, pulp chambers were implanted with biodegradable hydrogel-made scaffolds carrying MSCs together or without dermal microvascular ECs, and the cavities were sealed with mineral trioxide aggregate. After 14 days, pulp samples were analyzed by immunohistochemistry; messenger RNA expression of B-cell lymphoma 2 (Bcl-2), chemokine (C-X-C motif) ligand 1 (Cxcl1), CXC receptor 2 (Cxcr2), and dentin sialophosphoprotein (Dspp) by quantitative polymerase chain reaction, and protein expression of nestin and vascular endothelial growth factor by Western blotting. RESULTS: Teeth coimplanted with MSCs and ECs showed pulp healing with complete dentin bridge formation, whereas those implanted with MSCs alone had incomplete dentin bridges. Bcl-2, Cxcl1, Cxcr2, and Dspp messenger RNA levels were significantly up-regulated in the pulp of MSC/EC-implanted teeth compared with those in MSC-implanted teeth. Immunohistochemical analysis revealed the expression of nestin in odontoblastlike cells under dentin bridges in the MSC/EC coimplanted group. The density of CD31-expressing ECs and the expression of nestin and vascular endothelial growth factor proteins were significantly up-regulated in the MSC/EC-implanted pulp compared with the MSC-implanted pulp. CONCLUSIONS: The implantation of ECs with MSCs accelerated pulp tissue regeneration/healing and dentin bridge formation, up-regulated the expression of proangiogenic factors, and increased the density of ECs in pulpotomized rat molars.

Dental pulp tissue engineering of pulpotomized rat molars with bone marrow mesenchymal stem cells.

The major goal of dental pulp tissue engineering is to enable the healing of inflamed tissue or to replace necrotic pulp tissue with newly formed dental pulp tissue. Here, we report a protocol for pulp tissue engineering in vivo in pulpotomized rat teeth using constructs of rat bone marrow mesenchymal stem cells, preformed biodegradable scaffolds, and hydrogel. The constructs were implanted into pulpotomized pulp chambers for 3, 7, or 14 days. At 3 days, cells were located mainly along the preformed scaffolds. At 7 days, pulp tissue regeneration was observed in almost the entire implanted region. At 14 days, pulp tissue regeneration further progressed throughout the implanted region. In immunohistochemistry, at 3 days, a number of small and round macrophages immunoreactive to CD68 were predominantly distributed around the scaffolds. The density of CD68+ macrophages decreased until 14 days. On the other hand, nestin-expressing odontoblast-like cells beneath the dentin at the border of implanted region increased until 14 days. Quantitative gene expression analysis revealed that odontoblast differentiation marker dentin sialophosphoprotein mRNA in the implanted region gradually increased until 14 days. Together, the results suggested that regeneration of dental pulp tissue had occurred. Thus, our study provides a novel experimental rat model of dental pulp regeneration.

M2 macrophages participate in the biological tissue healing reaction to mineral trioxide aggregate.

INTRODUCTION: This study examined the protein and messenger RNA (mRNA) expression of molecules associated with M2 (wound healing) macrophages in mineral trioxide aggregate (MTA)-implanted rat subcutaneous tissue to elucidate the involvement of M2 macrophages in the connective tissue response to MTA. METHODS: Silicone tubes containing freshly mixed MTA or a calcium hydroxide cement (Life; Kerr, Romulus, MI) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. The specimens were then double immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, an M2 macrophage marker). Immunostaining for CD34 (a marker for vascularization and wound healing) was also performed. Expression levels of CD34, CD163, and mannose receptor c type 1 (an M2 macrophage marker) mRNAs were determined with real-time polymerase chain reaction. RESULTS: MTA-implanted subcutaneous tissues showed significant increases in the density of ED1+ED2+ macrophages beneath the implantation site and expression levels of CD163 and MMR mRNAs compared with Life-implanted and control tissues. MTA-implanted subcutaneous tissues also showed a significant increase of CD34-immunostained areas and up-regulation of CD34 mRNAs compared with Life-implanted and control tissues. CONCLUSIONS: MTA implantation induced the accumulation of M2 macrophage marker (ED2)-expressing macrophages and enhanced the expression of M2 macrophage marker genes. MTA implantation also enhanced the expression of CD34, suggesting acceleration of the healing/tissue repair process. Taken together, biological connective tissue response to MTA may involve wound healing/tissue repair processes involving M2 macrophages.

Evaluation of the responses of MHC class II molecule-expressing cells and macrophages to epoxy resin-based and 4-META-containing, methacrylate resin-based root canal sealers in rat subcutaneous tissue.

Major histocompatibility complex (MHC) class II molecule-expressing cells and macrophages play a pivotal role in mediating the host tissue response to biomaterials. This study investigated the responses of these cells to epoxy resin-based and 4-META-containing, methacrylate resin-based endodontic sealers (AH Plus and MetaSEAL respectively) in rat connective tissue. Silicone tubes loaded with one of the sealers or solid silicone rods (control) were subcutaneously implanted in male Wistar rats for three time periods of 7, 14, or 28 days. Tissue specimens were immunoperoxidase-stained for MHC class II molecules and CD68 (a general macrophage marker). Results showed that AH Plus-implanted tissue displayed significantly more MHC class II-positive cells than the control at 14 and 28 days, whereas MetaSEAL-implanted tissue showed significantly more CD68-positive cells than both AH Plus-implanted tissue and the control at all time periods. It was concluded that the epoxy resin-based sealer induced the infiltration of MHC class II molecule-expressing cells, whereas 4-META-containing, methacrylate resin-based sealer elicited macrophage infiltration.

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.

Immunohistochemical and gene expression analysis of stem-cell-associated markers in rat dental pulp.

Stem cells in the dental pulp comprise rare populations lacking definitive cytological markers and thus are poorly characterized in vivo, especially in rat species. To gain more insight into the phenotypical characteristics and tissue distribution of these cells, we examined the distribution of stem-cell-associated marker-expressing cells and mRNA expression levels of stem-cell-associated markers in the rat molar. CD146-positive cells co-expressing microtubule-associated protein 1B were counted following double-labeling immunoperoxidase staining and their density in the coronal pulp, root pulp and periodontal ligament was compared. Moreover, mRNA expression levels of CD146, CD105, CD166 and secreted phosphoprotein 1 (SPP1; also known as osteopontin, a negative regulatory element of the stem cell niche) were analyzed in these regions by using real time polymerase chain reaction. The double-positive cells could be clearly distinguished from non-stem cells single-stained by either of the markers and showed a significantly higher density in the coronal pulp compared with the other regions (P<0.05). Moreover, mRNA expression levels of CD146, CD105 and CD166 were significantly higher in the coronal pulp than in the other regions (P<0.05). On the other hand, SPP1 mRNA expression was significantly higher in the periodontal ligament than in the pulp. Thus, the density of stem-cell-associated marker-expressing cells and stem-cell-associated gene expression levels are higher in the coronal pulp than in the root pulp and periodontal ligament, suggesting that the coronal pulp harbors more stem cells than the other regions.

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.

Expression of angiogenic factors in rat periapical lesions.

INTRODUCTION: Angiogenic factors such as VEGFR2 (vascular endothelial cell growth factor receptor 2), Bcl-2 (a prosurvival and proangiogenic signaling molecule), and chemokine (C-X-C motif) ligand 1 (CXCL1) (a proangiogenic chemokine) may have critical roles in enhancing the establishment of apical periodontitis. To understand the role of these factors in the pathogenesis of apical periodontitis, we conducted immunohistochemical and molecular biological analysis. METHODS: Apical periodontitis was induced in the lower first molars of Wistar rats by making unsealed pulp exposures. After, 14, 21, and 28 days, the molars were retrieved, embedded as frozen sample blocks, and cut in a cryostat. Normal lower first molars served as controls. Immunostaining for CD31 (a marker for endothelial cells), Bcl-2, and real-time polymerase chain reaction analysis of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA were performed. In the real-time polymerase chain reaction analysis, messenger RNA was extracted from CD31-stained endothelial cells that were retrieved with laser capture microdissection. For statistical analysis of immunohistochemistry, the immunostained area was plotted, and pixel counts were determined. Then, the percentage of the immunostained area in the total area was calculated. RESULTS: The density of the CD31-stained area increased until 21 days after pulp exposure. On the other hand, Bcl-2-stained area showed the highest density at 14 days (active lesion expanding phase) and then decreased until 28 days (lesion stability phase). VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells showed the highest levels at 14 days and then decreased until 28 days. CONCLUSIONS: The increase in microvascular density and the up-regulation of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells took place coincidently with the expanding phase of experimentally induced periapical lesions. These data suggest that these angiogenic factors play a role in the lesion development.

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.

Laser-capture microdissection for Factor VIII-expressing endothelial cells in cancer tissues.

The immunostaining based Laser-capture microdissection (LCM) method called immune-LCM allows us to quantify the mRNA. Immune-LCM has recently been introduced to enhance identification of cells carrying a particular protein from frozen tissue samples. We have recently performed the immune-LCM of formaldehyde-fixated, paraffin-embedded tissues immunostained with a monoclonal antibody Factor VIII. This method could be useful for quantitative gene expression analysis in blood vessels from tumors of patients that have been treated with antiangiogenic drugs, allowing for validation of the effect of drug on the expected targets. Such capability might be exceedingly useful for the evaluation of the bioactivity of new drugs. This method is also useful to compare gene expression patterns in tumor cells versus endothelial cells during tumor progression or tumor angiogenesis.

Clinical management of dens invaginatus in a maxillary lateral incisor with the aid of cone-beam computed tomography--a case report.

This report describes non-surgical endodontic treatment of Oehlers' type III dens invaginatus in a maxillary lateral incisor with the aid of postobturation cone-beam computed tomography (CBCT). The endodontic treatment was initiated with the aid of a surgical operating microscope, and two canals, one of which represented the invagination, were instrumented, irrigated under passive ultrasonic activation and obturated with the lateral condensation technique. As postobturation periapical radiographs suggested the presence of untereated and/or unfilled areas in the root canal and invagination, CBCT was taken to assess the possibility of further treatment. The CBCT scans demonstrated inaccessible and unfilled canal and invagination areas because of complex internal morphology characterized by (i) C- or ring-shaped cross-sectional canal configuration with constrictions at different points in different root levels and (ii) a prominent intraradicular cavity that was communicated with the enamel-lined invagination and opened into the apical periodontium. Thus, it was judged that further endodontic treatment was not feasible. A 14-month follow-up revealed a satisfactory clinical and radiographic outcome, suggesting that the chemomechanical debridement may have sufficed to induce periapical healing. CBCT greatly helped the decision of avoiding further intervention that could have been difficult to negotiate.

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.