Initial transient accumulation of M2 macrophage-associated molecule-expressing cells after pulpotomy with mineral trioxide aggregate in rat molars.

INTRODUCTION: M2 (alternatively activated) macrophages are known to participate in wound healing and tissue repair. This study aimed to analyze the temporospatial changes in the distribution and density of M2 macrophage-associated molecule-expressing cells after pulpotomy with mineral trioxide aggregate (MTA) in rat molars to ascertain the role played by M2 macrophages in the healing of MTA-capped pulp tissue. METHODS: The maxillary first molars of 8-week-old Wistar rats were pulpotomized and capped with MTA. After 1-14 days, the teeth were examined after hematoxylin-eosin staining or immunoperoxidase staining of CD68 (a general macrophage marker) and M2 macrophage markers (CD163 and CD204). The density of positively stained cells was enumerated in the surface and inner regions (0-100 µm and 300-400 µm, respectively, from the wound surface). RESULTS: MTA capping initially caused mild inflammatory changes and the formation of a degenerative layer followed by progressive new matrix formation and calcified bridging. At 1-2 days, CD68-, CD163-, and CD204-positive cells started to accumulate beneath the degenerative layer, and the density of these cells was significantly higher in the surface region than in the inner region (P < .05). From 7 days onward, the 3 types of cells displayed an almost normal distribution beneath the newly formed dentinlike matrix. CONCLUSIONS: After the pulpotomy of rat molars with MTA, M2 macrophage-associated molecule-expressing cells transiently accumulated beneath the degenerative layer under the MTA. This suggests that M2 macrophages participate in the initial phases of the healing of MTA-capped pulp tissue.

Prostaglandin transporting protein-mediated prostaglandin E2 transport in lipopolysaccharide-inflamed rat dental pulp.

INTRODUCTION: The prostaglandin transporter (Pgt) and multidrug resistance-associated protein (Mrp) 4 are membrane transport proteins that play crucial roles in the transmembrane uptake and/or efflux of prostaglandins (PGs). This study attempted to analyze the protein expression of Pgt and Mrp4 and their involvement in PGE2 efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp tissue. METHODS: Pulpitis was induced in the upper incisors of Wistar rats by treating them with LPS for 24 hours. The protein expression levels of Pgt, Mrp4, and microsomal PGE synthase (mPGES) were analyzed with immunofluorescent staining. The amount of PGE2 released from the inflamed pulp tissue in the presence or absence of dipyridamole (an Mrp4 inhibitor) was assessed by using an enzyme-linked immunosorbent assay. RESULTS: Double immunofluorescence staining revealed that the Pgt, Mrp4, and mPGES immunoreactivity co-localized in CD31-expressing endothelial cells. Moreover, the Mrp4 inhibitor caused a significant decrease in the amount of PGE2 released from the LPS-inflamed pulp (P < .01 at 24 hours). CONCLUSION: Pgt, Mrp4, and mPGES expression was detected in the endothelial cells of normal and LPS-inflamed rat incisor pulp tissue, suggesting that these cells are associated with the biosynthesis and transmembrane transport of PGE2. The significant decrease in PGE2 release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE2 in the transmembrane efflux pathway.

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.

Odontoblast response to cavity preparation with Er:YAG laser in rat molars: an immunohistochemical study.

This study aimed to examine the dynamics of odontoblast-lineage cells following cavity preparation with erbium:yttrium-aluminum-garnet (Er:YAG) laser in rat molars. Cavity preparation was made with Er:YAG laser in the mesial surface of the maxillary left first molar of 8-week-old Wistar rats. Contralateral first molar served as unirradiated control. Immediately, 6 and 12 h and 1, 2, 3, 5 and 7 days after the lasing (n = 5, each), specimens were collected and processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin as markers for odontoblast-lineage cells. Cell proliferation assay using bromodeoxyuridine (BrdU) labeling was also performed. Unirradiated teeth showed HSP-25- and nestin-immunoreactivity in odontoblasts. At 6-12 h after irradiation, the odontoblastic layer was disorganized and some of odontoblasts lost the immunoreactivity to HSP-25 and nestin. At 1-2 days, however, HSP-25- and nestin-immunoreactivities in the odontoblast layer showed a noticeable recovery, resulting in the rearrangement of odontoblast-like cells intensely immunoreactive to HSP-25 and nestin at 3-7 days. BrdU-positive cells showed a significant increase at 2 days (P < 0.05 vs. immediate previous time point; one-way analysis of variance and Scheffé post hoc test), peaked at 3 days and then decreased significantly (P < 0.05). It was concluded that under the present experimental condition in rat molars, cavity preparation with Er:YAG laser induced mild and reversible damage to odontoblasts. The reparative process was characterized by the rearrangement of HSP-25- and nestin-immunoreactive odontoblast-like cells, which took place subsequent to the odontoblastic layer disorganization with partial loss of these immunoreactivities.

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp.

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)2 to induce a mineralized barrier at the exposed surface. After 7-42 days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42 days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.

Gene expression analysis of membrane transport proteins in normal and lipopolysaccharide-inflamed rat dental pulp.

INTRODUCTION: Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. METHODS: Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. RESULTS: The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). CONCLUSIONS: Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental 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.

Expressional alterations of fibrillin-1 during wound healing of human dental pulp.

INTRODUCTION: The degradation of fibrillins, the major constituents of microfibrils, is known to facilitate the release of active transforming growth factor-ß (TGF-ß), a signaling molecule contributing to mineralized tissue barrier formation in exposed dental pulps. To examine the involvement of fibrillins in the barrier formation, we examined the temporospatial expression of (1) genes and proteins of fibrillins and (2) factors possibly associated with fibrillin degradation and cytodifferentiation in exposed human pulps. Human pulp slice cultures were also examined for the role of fibrillins in mineralization. METHODS: Clinically healthy pulps were mechanically exposed and capped with mineral trioxide aggregate. After 7 to 42 days, the teeth were processed for immunohistochemical and cytochemical staining of fibrillin-1, fibrillin-2, latent TGF-ß-binding protein (LTBP)-1, matrix metalloproteinase-3 (MMP-3), alkaline phosphatase (ALP), and in situ hybridization of fibrillin-1. Pulp tissue slices cultured with ß-glycerophosphate were analyzed for fibrillin-1, fibrillin-2, and ALP with the immunohistochemical/cytochemical staining and quantitative reverse-transcriptase polymerase chain reaction. RESULTS: Fibrillin-1-immunoreactivity was seen until 7 days but turned into undetectable since 14 days in the pulpal area just beneath the exposure site. MMP-3-immunoreaction was transiently detected at 14 days. At 42 days when the mineralized barrier was evident, fibrillin-1-immunoreactivity and fibrillin-1 expression remained down-regulated. Fibrillin-2, LTBP-1, and ALP were constantly detected in the fibrillin-1-undetectable area. Pulp slices cultured with ß-glycerophosphate showed mineralization with up-regulation of ALP and down-regulation of fibrillin-1. CONCLUSIONS: Degradation and down-regulation of fibrillin-1 expression took place during the mineralized tissue barrier formation in exposed pulps in vivo and ß-glycerophosphate-induced pulpal mineralization in vitro.

GaAlAs laser irradiation induces active tertiary dentin formation after pulpal apoptosis and cell proliferation in rat molars.

INTRODUCTION: This study aimed to clarify pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation. METHODS: Maxillary first molars of 8-week-old rats were irradiated at an output power of 0.5 or 1.5 W for 180 seconds, and the samples were collected at intervals of 0 to 14 days. The demineralized paraffin sections were processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin in addition to cell proliferation assay using bromodeoxyuridine (BrdU) labeling and apoptosis assay using deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL). RESULTS: Intense HSP-25 and nestin immunoreactivities in the odontoblast layer were weakened immediately after 0.5-W irradiation and recovered on day 1, resulting in slight tertiary dentin formation by day 14. On the contrary, 1.5-W irradiation immediately induced the loss of HSP-25 and nestin-immunoreactivities in the odontoblast layer. On day 1, numerous TUNEL-positive cells appeared in a degenerative zone that was surrounded by intense HSP-25 immunoreactivity. BrdU-positive cells occurred within the intensely HSP-25-immunopositive areas during days 2 through 5, whereas TUNEL-positive cells gradually decreased in number by day 5. HSP-25- and nestin-positive odontoblast-like cells were arranged along the pulp-dentin border by day 7, resulting in remarkable tertiary dentin formation on day 14. CONCLUSIONS: The output energy determined pulpal healing patterns after GaAlAs laser irradiation; the higher energy induced the apoptosis in the affected dental pulp including odontoblasts followed by active cell proliferation in the intense HSP-25-immunoreactive areas surrounding the degenerative tissue, resulting in abundant tertiary dentin formation. Thus, the optimal GaAlAs laser irradiation elicited intentional tertiary dentin formation in the dental pulp.

Immunohistochemical analysis of nestin, osteopontin, and proliferating cells in the reparative process of exposed dental pulp capped with mineral trioxide aggregate.

This study investigated the reparative process of mechanically exposed pulps capped with mineral trioxide aggregate (MTA). Maxillary first molars of 8-week-old rats were MTA-capped for 1-14 days, and 5-bromo-2'-deoxyuridine-labeled proliferating cells and immunoreactivity for nestin and osteopontin were analyzed. MTA capping caused mild necrotic changes followed by progressive new matrix formation and calcified bridging. Proliferating cells peaked at 3 days when matrix formation was inconspicuous. Nestin-expressing cells appeared at 3 days, were arranged beneath the newly formed matrix at 5 days, and showed odontoblast-like morphology by 14 days. Osteopontin immunoreactivity was detected just beneath the necrotic area after 1 day. These findings suggest that pulpal responses to MTA capping involve proliferation and migration of progenitors followed by their differentiation into odontoblast-like cells, a mechanism basically similar to those to calcium hydroxide. Osteopontin might play a triggering role in initiation of the pulpal reparative process.

Impact of Streptococcus mutans on the generation of fluorescence from artificially induced enamel and dentin carious lesions in vitro.

The purpose of this study was to examine whether Streptococcus mutans is implicated in the generation of fluorescence detected in carious lesions. Enamel surfaces and dentin cavities of extracted human teeth were subjected to artificial caries generation by exposing them either to a culture medium containing S. mutans or to a lactic acid buffer for 2 weeks. Fluorescence from the lesions was detected with confocal laser scanning microscopy or fluorescence microscopy at various excitation wavelengths, and maximum fluorescence radiance was computed using imageanalyzing software. Culture media of S. mutans were also examined for fluorescence generation. The results demonstrated that S. mutans-induced enamel and dentin lesions exhibited increased fluorescence in the red and green spectral regions, with the signal stronger in the red region. In the blue region, however, fluorescence signals in the corresponding area were below the background level. Significantly weaker or virtually no fluorescence was detected in lactic acid-demineralized lesions at all excitation wavelengths. Neither bacterial cells nor culture media generated any fluorescence. These results indicate that, although the presence of S. mutans may be a prerequisite for the emission of fluorescence from carious lesions, some interaction of S. mutans with exposed tooth matrix elements may also be required for the generation or unmasking of fluorophores.

All-in-one self-etch model adhesives: HEMA-free and without phase separation.

OBJECTIVE: Aim of this study was to design HEMA-free all-in-one self-etch model adhesives without phase separation, and to investigate their efficiency on extracted human teeth. MATERIALS AND METHODS: Compositions of adhesives in mass% (1): UDMA (25), 4-META (20), H(2)O (0, 1, 2, 5, 10, 20, 35, and 45), balance of acetone or ethanol. (2): UDMA (35), 4-META or 4-MET (28), H(2)O (0, 2, 4, 5, 6, and 8), balance of acetone. Phase separation was evaluated on samples exposed to ambient atmosphere. Conventional shear bond strengths (SBS, n=8) were determined on human enamel and dentin. Marginal adaptation (MGW, n=8) was assessed in cylindrical butt-joint dentin cavities. RESULTS: Solutions (1) and (2) with 5 and 8% or less water content, respectively, showed no phase separation. SBSs on enamel were not different within the acetone- or ethanol-group and between the adhesive groups (1). Water content of adhesives (2) was a significant determinant of enamel SBSs, groups with 4-META or 4-MET were not different (p>0.05). Dentin SBSs with adhesives (1) were not different (p>0.05) within solvent groups, yet higher for acetone-dissolved adhesives (p<0.05). Dentin SBSs with adhesives (2) were different by water content and functional monomer (p<0.05). MGW for solutions (1) were smaller with acetone-dissolved than with ethanol-dissolved adhesives (p<0.001). Acetone solutions between 2 and 45% water content produced almost perfect marginal adaptation. Marginal adaptation of adhesives (2) was almost perfect at 5 through 8% water content. CONCLUSIONS: Simplified HEMA-free self-etch adhesives without phase separation were prepared without compromises on bonding efficiency to enamel and dentin.

Removal of resin-based root canal filling materials with K3 rotary instruments: relative efficacy for different combinations of filling materials.

Removal of resin-based root canal filling materials may cause serious problems during root canal retreatment. This study compared the working time and amount of canal enlargement when different resin-based root canal filling materials were removed with K3 rotary instruments with or without heat-softening using System B. Root canal sealer/filling point combinations tested were Epiphany/Resilon, SuperBond/Resilon, SuperBond/gutta-percha, and Canals N/gutta-percha. The materials were filled into simulated curved resin canals and removed with K3 instruments in a standardized crown-down procedure. In terms of working time, Epiphany/Resilon required a significantly longer working time than the others. However, heat application with System B significantly reduced the working time for the removal of Epiphany/Resilon. In terms of canal enlargement, there were no significant differences among the tested groups as determined with digital morphometry. It was thus concluded that Epiphany removal with K3 rotary instruments might result in extended working time, but which could be reduced with heat-softening using System B.

Caries diagnosis using a laser fluorescence system--observation of autofluorescence of dental caries--.

The purpose of this study was to evaluate the usefulness of autofluorescence of carious lesions on caries diagnosis. The observation of the micromorophology of caries lesions was conducted using a confocal laser scanning microscope, a fluorescence microscope and a WDX type electron probe X-ray microanalyzer. Observation of autofluorescence under Cy5 and UV fields showed clearly specific images of autofluorescence in the carious lesion. However, observation of autofluorescence under FITC field showed images of autofluorescence with unclear boundaries in the carious lesion. EPMA images showed decreases in Ca and P in the carious areas. As a result of the observation of autofluorescence and the EPMA images in the carious lesion, a correlation was noted between autofluorescence under the Cy5 field as the laser fluorescence apparatus for caries diagnosis and demineralized areas. The usefulness of autofluorescence of carious lesion on caries diagnosis was suggested.

A study of cavity preparation by Er:YAG laser. Effects on the marginal leakage of composite resin restoration.

The purpose of this study was to evaluate marginal leakage of composite resin restoration from cavities prepared by Er:YAG laser. The observation of the dentin surface after the application of laser irradiation was performed by LSM, the cutting surface showed a rough surface similar to scales, and exposed dentinal tubules were observed without striations or a smeared layer formation that were observed when using a rotary cutting device. Leakage tests revealed no significant differences in the marginal seal for both enamel and dentin between cavities prepared by Er:YAG laser irradiation and when using an air-turbine. In this study, the usefulness of cavity preparation by Er:YAG laser irradiation in composite resin restoration was suggested.

A study of cavity preparation by Er:YAG laser--observation of hard tooth structures by laser scanning microscope and examination of the time necessary to remove caries.

The purpose of this study was to observe and measure the morphological changes that occur in the hard tissue after the application of Er:YAG laser. Another objective was to evaluate and compare the duration of application of both the laser apparatus and a conventional cutting device. In this study, sound and newly extracted carious tissues were used. The morphological changes in hard tooth structures produced by Er:YAG laser irradiation were examined by using a laser scanning microscope. Results showed that appropriate laser irradiation was 100 mJ/pulse for dentin, and 200 mJ/pulse for enamel. Also, the laser scanning microscope images were less damaged than the SEM images due to pretreatment of the specimens. The time taken to remove carious enamel by laser irradiation was slightly longer than the compared rotary cutting device; however, no differences between the two methods were observed in case of carious dentin removal.