Yusuke Yamanaka

Immunohistochemical analysis of subcutaneous tissue reactions to methacrylate resin-based root canal sealers

AIM To investigate subcutaneous tissue reactions to methacrylate resin-based root canal sealers by immunohistochemical assessment of inflammatory/immunocompetent cell infiltration. METHODOLOGY Silicone tubes containing freshly mixed Epiphany SE sealer, MetaSEAL, Super-Bond RC sealer, or a zinc oxide-eugenol sealer (Canals) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. After 7, 14 and 28 days, connective tissue surrounding the implants (n = 8, each) was processed for immunoperoxidase staining using OX6 (reactive to major histocompatibility complex class II molecules), ED1 (reactive to macrophages), and W3/13 (reactive primarily to neutrophils), and the number of positively stained cells within each field (1.2 × 0.8 mm) was enumerated. Statistical differences were analysed with Friedmans test and Scheffes test (comparisons between test materials) or Mann-Whitneys U-test (test-control comparisons). RESULTS Canals showed a significantly higher number of W3/13-positive cells (mostly neutrophils) than MetaSEAL at 28 days (P < 0.05). There were no significant differences in the numbers of OX6- or ED1-positive cells between each test material at any time point. Test-control comparisons revealed several significant differences for each antibody. This was most notable for ED1, where all the test materials at each time point, except for Epiphany SE at 28 days, showed significantly larger values than the corresponding controls. CONCLUSIONS All the methacrylate resin-based sealers tested showed a similar level of inflammatory/immunocompetent cell infiltration. MetaSEAL induced less-intense neutrophil infiltration than Canals. Controls exhibited milder infiltration of inflammatory/immunocompetent cells compared with all the test materials.

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.

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.

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.

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. Microsc. Res. Tech. 2012.