Tomoatsu Kaneko

Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHED) have been isolated and characterized as multipotent cells. However, it is not known whether SHED can generate a dental pulp-like tissue in vivo. The purpose of this study was to evaluate morphologic characteristics of the tissue formed when SHED seeded in biodegradable scaffolds prepared within human tooth slices are transplanted into immunodeficient mice. We observed that the resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp. Ultrastructural analysis with transmission electron microscopy and immunohistochemistry for dentin sialoprotein suggested that SHED differentiated into odontoblast-like cells in vivo. Notably, SHED also differentiated into endothelial-like cells, as demonstrated by B-galactosidase staining of cells lining the walls of blood-containing vessels in tissues engineered with SHED stably transduced with LacZ. This work suggests that exfoliated deciduous teeth constitute a viable source of stem cells for dental pulp tissue engineering.

Bcl-2 Acts in a Proangiogenic Signaling Pathway through Nuclear Factor-κB and CXC Chemokines

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.

Ultrastructural analysis of MHC class II molecule-expressing cells in experimentally induced periapical lesions in the rat.

Periapical lesions were induced by making 28 days of unsealed pulp exposures in the lower first molars of Wistar rats. Major histocompatibility complex class II molecule-expressing cells were then demonstrated by means of immunoperoxidase staining using a monoclonal antibody OX6, and the ultrastructure of these cells was analyzed under electron microscopy. OX6+ cells were classified into two major populations, (i.e. macrophages and dendritic cell (DC)-like cells. DC-like cells had elongated cytoplasmic processes, contained a few lysosomal structures, lacked distinct phagosomes, and were the most predominant cell type in the established lesion. Some of lymphocytes and plasma cells also showed a positive immunoreactivity. Both OX6+ macrophages and DC-like cells often showed a cell-to-cell attachment with lymphocytes. These findings suggested that major histocompatibility complex class 11 molecule-expressing macrophages and DC-like cells may play a crucial role in periapical lesion development by acting as antigen-presenting cells to memory T lymphocytes.

Immunocompetent cells in the pulp of human deciduous teeth

This immunohistological study sought to determine how the distribution and density of various immunocompetent cells change in the pulp of human deciduous teeth during the process of physiological root resorption. Forty-three extracted deciduous teeth at various stages of resorption were subjected to immunoperoxidase staining with the use of antibodies directed to HLA-DR, CD68, factor XIIIa and lymphocyte subsets. In intact deciduous teeth (group 0), all types of cells examined, except for CD20+ B lymphocytes, were detected. In teeth in which resorption was less than 1/3 of the root length (group 1), all types of cells showed a statistically significant increase compared with group 0 (P<0.05; Mann-Whitneys U-test). HLA-DR+, CD68+, and factor XIIIa+ cells with a dendritic profile kept their distribution in the periphery of the pulp, and oval and round, newly recruited macrophages accumulated in the central portion of the pulp and near the resorption sites. In teeth where resorption was 1/2 to 2/3 (group 2), all the cell types increased further. Aggregations of HLA-DR+, CD68+, and factor XIIIa+ cells were frequently seen in the central portion of the pulp, and T and B lymphocytes occasionally formed some clusters. Comparisons with group 1 revealed that the density of these cells, except for CD20+ cells, showed significant increases (P<0.05; Mann-Whitneys U-test). These results provided evidence showing that immunocompetent cells of deciduous tooth pulp increase with the progress of physiological root resorption, suggesting that immunocompetency of deciduous teeth is altered by this process.

An immunoelectron-microscopic study of class II major histocompatibility complex molecule-expressing macrophages and dendritic cells in experimental rat periapical lesions

Previous studies have demonstrated that heterogeneous populations of class II major histocompatibility complex (MHC) molecule-expressing non-lymphoid cells, ultrastructurally classified as macrophages and dendritic cell (DC) cell-like cells, comprise the major immune cell population in experimental periapical lesions in rat molars. In this study, the temporal changes in relative proportions of the two types of cells were examined, on the hypothesis that they are involved in different aspects of the pathogenesis of the lesions. The lesions were induced by making surgical pulp exposures in mandibular first molars of 5-week-old Wistar rats. Observation periods were set at 0 (normal), 3, 14, 28, and 56 days. Non-lymphoid cells immunoreactive to OX6 (reactive to class II MHC molecules) were classified as macrophages and DC cell-like cells according to their ultrastructure, and the frequencies of the two types of cells were assessed at each time-point. ED1 (reactive to nearly all macrophages and DCs) was also used to identify macrophages and DC cell-like cells. At 3 days, most OX6+ cells and ED1+ cells in the periapical tissue had the ultrastructural appearance of newly recruited macrophages. At 14 days, when the lesion was actively expanding, there were significantly more OX6+ macrophages than OX6+ DC cell-like cells (P<0.01). However, at 28 days, when lesion expansion had ceased, DC cell-like cells significantly outnumbered OX6+ macrophages (P<0.01); this remained constant at 56 days. Cell-to-cell contact between OX6+ non-lymphoid cells and OX6- lymphocytes, suggesting a functional interaction, was most frequently seen at 28 days. These results support the notion that class II MHC molecule-expressing macrophages play some part in the initial lesion expansion, and suggest that DC cell-like cells may primarily be involved in immune defence against perpetuated antigenic challenges following lesion stabilization.

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP‐F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained (P < 0.05). Correlations were obtained between the CENP‐F LI and tumor size (P < 0.05). Immunoelectron microscopy showed CENP‐F nuclear staining as punctate or fine dots. The present study shows that CENP‐F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G2 to M cells, compared to Ki‐67.

Localization and density of myeloid leucocytes in the periodontal ligament of normal rat molars

The phenotypic distribution and density of macrophage-associated antigen-expressing cells in the periodontal ligament (PDL) of normal rat mandibular first molars was evaluated by immunohistochemistry, and an attempt made to identify dendritic cells (DCs) by immunoelectron microscopy. Cells immunopositive to ED1 (a general macrophage marker) were widely distributed throughout the PDL and were most common around blood vessels. A small number of T lymphocytes and OX62 (anti-veiled cells and gammadelta T cells)-positive DC-like cells were also found. The relative density of cells immunopositive to ED9 (CD14), OX42 (CD11b), OX6 (anti-class II MHC molecules), ED2 (anti-tissue-resident macrophages), 8A2 (CD11c) and WT.1 (CD11a) varied in the mesial, distal and periapical regions of the distal root and the furcal region. This finding suggests that there are several subpopulations of ED1-positive cells which express various combinations of these markers. Immunoelectron microscopy revealed that a small, but distinct, subpopulation of ED1- and OX6-positive cells did have a DC-like ultrastructure, although the majority of these cells were identified as macrophages. The DC-like cells were characterized by poorly developed lysosomal structures and an absence of phagocytic vesicles. It was concluded that the normal rat PDL is equipped with heterogeneous populations of macrophages with regional variations in density. The DC-like cells may function as antigen-presenting cells.

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 Immunostained Endothelial Cells Isolated From Formaldehyde-Fixated Paraffin Embedded Tumors Using Laser Capture Microdissection—A Technical Report

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde‐fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof‐of‐principle study, we analyzed by reverse transcription‐PCR (RT‐PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl‐2 in the endothelial cells was evaluated by RT‐PCR and by real time PCR. Glyceraldehyde‐3‐phosphate dehydrogenase and 18S were used as house keeping genes for RT‐PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. Microsc. Res. Tech., 2009.