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Hydroxyapatite formation on endodontic hydraulic calcium silicate cements (HCSCs) plays a significant role in sealing the root canal system and elevating the hard-tissue inductivity of the materials. This study aimed to evaluate the in vivo apatite-forming ability of 13 second-generation HCSCs using a representative first-generation HCSC (white ProRoot MTA: PR) as a positive control. Thirteen second-generation HCSCs and PR were loaded into polytetrafluoroethylene tubes and implanted in subcutaneous tissue of 4-week-old male Wistar rats. At 28 days after implantation, hydroxyapatite formation on the HCSC implants was assessed with micro-Raman spectroscopy, surface ultrastructural and elemental characterization, and elemental mapping of the material–tissue interface. A Raman band for hydroxyapatite (v1 PO43- band at 960 cm−t) and hydroxyapatite-like calcium-phosphorus-rich spherical precipitates were detected on six second-generation HCSCs and PR. In the elemental mapping, calcium-phosphorus-rich hydroxyapatite-layer-like regions were not observed on the seven HCSCs that showed neither the hydroxyapatite Raman band nor hydroxyapatite-like spherical precipitates. These results indicated that only 6 of the 13 second-generation HCSCs produced a detectable amount of hydroxyapatite in rat subcutaneous tissue within 28 days, similar to PR. The seven second-generation HCSCs that did not exhibit hydroxyapatite formation may not be suitable alternatives to PR due to their weak in vivo apatite-forming ability.

Ascorbic acid (AA; vitamin C) plays a crucial role in the biosynthesis and secretion of collagen to produce the organic matrix of hard tissues. Nevertheless, the detailed mechanism by which AA induces reparative dentinogenesis is still unknown. This study aimed to investigate the pathway and function of AA during wound healing in a rat pulpotomy model. Sodium-dependent vitamin C transporter (SVCT) 2 and glucose transporter (GLUT) 1 were detected in odontoblasts, endothelial cells, and nerve fibers in normal pulp tissues. SVCT2 and GLUT1 were also expressed in odontoblast-like cells in pulpotomized tissues of Wistar rats and osteogenic disorder Shionogi (ODS) rats, which cannot generate AA. However, in ODS rats, a thick layer of osteopontin was detected beneath the wound surface, and odontoblast-like cells observed along this layer expressed Nestin and α-SMA, but the formation of dentin bridges was not evident. Macrophages expressing CD68 and CD206 increased beneath the wound site. Hence, AA may be involved in odontoblast-like cell differentiation and anti-inflammatory response during dental pulp wound healing. Our results provide new insights into the function of AA through SVCT2 and GLUT1 in reparative dentinogenesis and may help in developing new therapeutic targets for dental pulpal disease.

Objectives The objective of this study was to evaluate the in vivo apatite-forming ability of 17 second-generation hydraulic calcium silicate cements (HCSCs) with the first-generation HCSC, white ProRoot MTA (PR). Materials and Methods Seventeen second-generation HCSCs and PR were implanted in rat subcutaneous tissue for 28 days. After inplantation, Raman spectra were taken from the surface of the HCSC implants and blindly evaluated for the presence or absence of a band at 960 cm− 1 indicating apatite. Apatite formation was also assessed with surface characterization and elemental mapping. Results The Raman band for apatite was detected on only seven second-generation HCSCs and PR. These seven HCSCs exhibited apatite-like calcium- and phosphorus-rich spherical precipitates on their surface. Three types of HCSCs had a Raman band at 962 cm− 1 that may have obscured the Raman band for apatite at 960 cm− 1; however, elemental mapping demonstrated the absence of calcium-phosphorus-rich apatite-layer-like regions on these HCSCs. Conclusions Only 7 of the 17 second-generation HCSCs and PR produced apatite in vivo within 28 days. Clinical Relevance: The 10 types of second-generation HCSCs that did not exhibit apatite formation may not be suitable substitutes for PR due to their weak in vivo apatite-forming ability.

Objectives To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. Subjects and methods Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. Results Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. Conclusions After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2–coated plates upregulated CD163 expression, compared with laminin-511/E8-α5–coated plates. Integrin α3– and integrin α6–neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin–laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Resin monomers and polymerisation initiators have been shown to be cytotoxic for pulp cells and to disturb odontoblast differentiation. This study aimed to compare the effect of a resin-modified calcium silicate cement (TheraCal LC; TC) and a resin-free calcium silicate cement (ProRoot MTA; PR) on pulpal healing after pulpotomy. Pulpotomy was performed on the maxillary first molars of 8-week-old rats using either PR or TC. After 1, 3, 7, 14 and 28 days, pulpal responses were assessed by micro-computed tomography, haematoxylin-eosin staining and immunostaining against CD68, which is a pan-macrophage marker. The results showed that pulpotomy with TC induced persistent infiltration of inflammatory cells, including CD68-positive macrophages, and delayed the formation of reparative dentin as compared with that with PR, although both materials allowed pulpal healing over the long term. Therefore, resin-modified TC was not as biocompatible nor bioinductive as resin-free PR when applied on the healthy pulp of rat molars.

This study compared the apatite-forming ability (AFA) levels of flowable and putty formulations of Nishika Canal Sealer BG Multi (F-NBG and P-NBG, respectively) and attempted to clarify the cause of differences in the AFA levels of F-NBG and P-NBG. NBG samples were aged in simulated body fluid (SBF) or 1-, 5-, or 10-g/L bovine serum albumin-containing SBF (BSA-SBF) and analyzed in terms of their ultrastructures, elemental compositions, and Raman spectra to identify apatite formation. The phosphate ion consumption rates of NBG samples in the media were evaluated as an indicator of apatite growth. The original elemental composition, calcium ion release, and alkalizing ability levels of F-NBG and P-NBG were also evaluated. Apparent apatite formation was detected on all NBG samples except F-NBG aged in 10-g/L BSA-SBF. P-NBG consumed phosphate ions faster than F-NBG. As-prepared P-NBG showed more silicon elements on its surface than as-prepared F-NBG. P-NBG released more calcium ions than F-NBG, although their alkalizing ability levels did not differ statistically. In conclusion, the AFA of P-NBG was greater than that of F-NBG, probably because of the greater ability of P-NBG to expose silanol groups on the surface and release calcium ions.

Bioceramic-containing root canal sealers promote periapical healing via Ca2+ and OH− release and apatite formation on the surface. This study aimed to compare Ca2+ and OH− release and in vivo apatite formation of three bioceramic-containing root canal sealers: EndoSequence BC sealer (Endo-BC), MTA Fillapex (MTA-F), and Nishika Canal Sealer BG (N-BG). Polytetrafluoroethylene tubes filled with sealers were immersed in distilled water for 6 and 12 h and for 1, 7, 14, and 28 days to measure Ca2+ and OH− release. Additionally, tubes filled with sealers were implanted in the backs of rats for 28 days, and in vivo apatite formation was analyzed using an electron probe microanalyzer. Endo-BC released significantly more Ca2+ than the other sealers at 6 and 12 h and 1 day. Ca2+ release was significantly lower from N-BG than from Endo-BC and MTA-F at 14 and 28 days. OH− release was significantly higher from Endo-BC than from the other sealers throughout the experiment, except at 1 day. OH− release was lower from N-BG than from MTA-F at 6 h and 7 days. Only Endo-BC implants exhibited apatite-like calcium-, phosphorus-, oxygen-, and carbon-rich spherulites and apatite layer–like calcium- and phosphorus-rich, but radiopaque element-free, surface regions. Ca2+ and OH− release is ranked as follows: Endo-BC > MTA-F > N-BG. Only Endo-BC demonstrated in vivo apatite formation. Endo-BC could promote faster periapical healing than MTA-F and N-BG.

When regenerative endodontic procedures (REPs) are performed on immature teeth diagnosed with pulp necrosis and apical periodontitis, various healing patterns occur. Furthermore, infected immature teeth with endodontic disorders often exhibit some remnant pulp and apical tissue. Therefore, this study investigated the impact of remnant healthy or fully functional pulp and apical tissue on healing patterns after REPs. Simulated REPs were performed on non-infected immature rat molars with different amounts of remnant pulp and apical tissue. Healing patterns in these teeth were assessed after 28 days. Teeth with 0.81–0.91 mm of remnant pulp healed with pulp-like tissue, dentin, and osteodentin-like dentin-associated mineralized tissue (OSD-DAMT); teeth with 0.60–0.63 mm of remnant pulp healed with pulp-like tissue and OSD-DAMT; teeth with 0.13–0.43 mm of remnant pulp healed with periodontal ligament (PDL)-like tissue, OSD-DAMT, and cementum-like dentin-associated mineralized tissue (CEM-DAMT); and teeth with disorganization of pulp and apical tissues at 0.15–0.38 mm beyond the root apex healed with PDL-like tissue, CEM-DAMT, and intracanal bone (IB). Loss of Hertwig’s epithelial root sheath was observed with IB formation. These results showed that four distinct healing patterns occurred after REPs, depending on the preoperative amount of remnant healthy pulp and apical tissue.

AIM To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue. METHODOLOGY A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test. RESULTS In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days. CONCLUSIONS The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation.

Aim To evaluate the abilities of three calcium silicate-based pulp-capping materials (ProRoot MTA, TheraCal LC, and a prototype tricalcium silicate cement) to produce apatite-like precipitates after being subcutaneously implanted into rats. Methodology Polytetrafluoroethylene tubes containing each material were subcutaneously implanted into the backs of Wistar rats. At 7, 14, and 28 days post-implantation, the implants were removed together with the surrounding connective tissue, and fixed in 2.5% glutaraldehyde in cacodylate buffer. The chemical compositions of the surface precipitates formed on the implants were analyzed with scanning electron microscopy-electron probe microanalysis (SEM-EPMA). The distributions of calcium (Ca) and phosphorus (P) at the material-tissue interface were also analyzed with SEM-EPMA. Comparisons of the thicknesses of the Ca- and P-rich areas were performed using Friedman test followed by Scheffe's test at a significant level of 5%. Results All three materials produced apatite-like surface precipitates containing Ca and P. For each material, elemental mapping detected a region of connective tissue in which the concentrations of Ca and P were higher than those seen in the surrounding connective tissue. The thickness of this Ca- and P-rich region exhibited the following pattern: ProRoot MTA > prototype tricalcium silicate cement ≥ TheraCal LC. ProRoot MTA had a significantly thicker layer of Ca and P than the other materials at all time points (P < 0.05), and a significant difference was detected between the prototype cement and TheraCal LC at 28 days (P < 0.05). Conclusions After being subcutaneously implanted, all of the materials produced Ca- and P-containing surface precipitates and a Ca- and P-rich layer within the surrounding tissue. The thickness of the Ca- and P-rich layer of ProRoot MTA was significantly thicker than that of the other materials. This article is protected by copyright. All rights reserved.

Introduction Myofibroblasts express alpha smooth muscle actin (α-SMA) and play a critical role in wound healing. Myofibroblast differentiation is controlled by the joint actions of transforming growth factor beta 1 (TGF-β1) and the extradomain A fibronectin splice variant (EDA-FN). Currently, the contribution of myofibroblasts to dental pulp healing is unknown. Therefore, we analyzed expressional characteristics of α-SMA–positive cells and investigated TGF-β1, EDA-FN, and α-SMA expression levels after pulpotomy to better understand dental pulp healing. Methods The maxillary first molars of 8-week-old Wistar rats were pulpotomized with mineral trioxide aggregate. After 1 to 14 days, localization and colocalization of α-SMA, rat endothelial cell antigen-1 (as a marker of endothelial cells), neuron-glial antigen 2 (as a marker of perivascular cells), prolyl-4-hydroxylase (P4H, as an additional marker of myofibroblasts), and EDA-FN were analyzed using immunohistochemistry and double immunofluorescence. Time-course changes in the messenger RNA expression levels of TGF-β1, EDA-FN, and α-SMA were evaluated using quantitative real-time polymerase chain reaction analysis. Results Spindle-shaped α-SMA–positive cells transiently appeared after pulpotomy. These cells initially emerged in the pulp core on day 3 and then accumulated at the wound site by day 5. These cells were isolated from rat endothelial cell antigen-1 positive cells and did not express neuron-glial antigen 2 but did express P4H. The messenger RNA levels of TGF-β1, EDA-FN, and α-SMA were significantly up-regulated after pulpotomy. EDA-FN and α-SMA were colocalized at the wound sites on day 5. Conclusions In association with up-regulation of TGF-β1 and EDA-FN expression, α-SMA and P4H double-positive cells accumulated at the wound sites after pulpotomy. This suggests that myofibroblasts participate in dental pulp healing.

This study aimed to analyze the mRNA expression and protein localization of prostaglandin I2 (PGI2) synthase (PGIS), the PGI2 receptor (IP receptor) and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) in force-stimulated rat molars, toward the elucidation of the PGI2-IP receptor-TRPV1 pathway that is in operation in the pulp and possibly associated with orthodontic pain and inflammation. Experimental force was applied to the maxillary first and second molars by inserting an elastic band between them for 6–72 h. PGIS, PTGIR (the IP receptor gene), and TRPV1 mRNA levels in the coronal pulp were analyzed with real-time PCR. PGIS, IP receptor, and TRPV1 proteins were immunostained. The force stimulation induced significant upregulation of PGIS at 6–24 h, and PTGIR and TRPV1 at 6 and 12 h in the pulp. PGIS was immunolocalized in odontoblasts and some fibroblasts in the force-stimulated pulp. The IP receptor and TRPV1 immunoreactivities were detected on odontoblasts and some nerve fibers. It was concluded that PGIS, PTGIR, and TRPV1 in rat molar pulp were significantly upregulated shortly after the force application, and that the IP receptor was co-expressed on TRPV1-expressing nerves and odontoblasts. These findings suggest that the PGI2-IP receptor-TRPV1 pathway is associated with the acute phase of force-induced pulp changes involving odontoblasts and nerves.

Introduction Pulp capping materials allow healing of injured pulp with a layer of reparative dentin. Glucose is needed to cure the injured area. Glucose is transported by glucose transporter (Glut) 2 and Glut4, which are transmembrane proteins that act as gatekeepers. We hypothesized that the transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing. Therefore, we explored Glut2 and Glut4 expression during reparative dentinogenesis after mineral trioxide aggregate capping. Methods The upper left first molar of 8-week-old Wistar rats underwent pulpotomy with mineral trioxide aggregate. At 1, 3, 5, 7, and 14 days after treatment, localization and colocalization of Glut2, Glut4, nestin (odontoblast marker), and antiendothelial cell antigen 1 (RECA-1; endothelial cell marker) were analyzed with immunohistochemical staining. Messenger RNA expression levels of Slc2a2 (encoding Glut2), Slc2a4 (encoding Glut4), Igf-1r (encoding insulinlike growth factor 1 receptor), and nestin were analyzed in the extracted teeth using real-time polymerase chain reaction. Results Glut2 and Glut4 were localized within odontoblasts and endothelial cells in normal control teeth. Three days after pulpotomy, Glut2- and Glut4-positive cells were detected; 7 days after pulpotomy, immunoreactivity for Glut2 and Glut4 was confined to newly differentiated odontoblastlike cells arranged beneath reparative dentin. Messenger RNA expression levels of Slc2a2 and Slc2a4 were significantly up-regulated after pulpotomy. Conclusions Glut2 and Glut4 regulate glucose transport during wound healing beneath the injured area. This may contribute to the development of new vital pulp therapy for patients with deep caries.

Aim To examine the temporospatial expression of dentine matrix protein 1 (DMP1; a noncollagenous protein involved in mineralized tissue formation), osteopontin (another noncollagenous protein detected during reparative dentinogenesis) and nestin (a marker of differentiating/differentiated odontoblasts), following direct pulp capping with calcium hydroxide in rat molars. Methodology The maxillary first molars of 8-week-old Wistar rats had their pulps exposed and capped with calcium hydroxide. The pulp-capped teeth were collected from 6 h to 14 days postoperatively and processed for immunohistochemistry for DMP1, osteopontin and nestin. Cell proliferation was monitored using 5-bromo-2′-deoxyuridine (BrdU) labelling. Results The capped pulps initially exhibited superficial necrotic changes followed by the formation of new matrix and its mineralization. DMP1 immunoreactivity was observed in the matrix beneath the necrotic layer from 6 h onwards and present in the outer portion of the newly formed mineralized matrix from 7 days onwards. Osteopontin displayed a similar expression pattern, although it occupied a narrower area than DMP1 at 6 and 12 h. Nestin-immunoreactive cells appeared beneath the DMP1-immunoreactive area at 1 day, were distributed beneath the newly formed matrix at 5 days and exhibited odontoblast-like morphology by 14 days. BrdU-positive cells significantly increased at 2 and 3 days (P

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

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.

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 Oatp1a5 , Oatp2b1 ( P and Oatp3a1 ( P 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.

Yamanaka Y, Shigetani Y, Yoshiba K, Yoshiba N, Okiji T. Immunohistochemical analysis of subcutaneous tissue reactions to methacrylate resin-based root canal sealers. International Endodontic Journal, 44, 669–675, 2011. Abstract 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 Friedman’s test and Scheffe’s test (comparisons between test materials) or Mann–Whitney’s 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

Regeneration of periodontal tissues, lost as a result of periodontal disease, is a key objective of periodontal treatment. Although several periodontal regeneration therapies have been devised, the origin of the undifferentiated cells that regenerate periodontal tissues remains unknown. Therefore, in the present study, to clarify the existence of osteoblast progenitor cells in the periodontal ligament, as well as to investigate the mechanism of alveolar bone regeneration without any effects from the original bone, we evaluated osteoblast differentiation induced by transplantation of GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Ten days after transplantation, initial alveolar bone was formed apart from the cementum in the bifurcation region. After 20 days, this bone tissue had expanded to almost all of the bifurcation. GFP localization showed that the osteoblasts were derived from the transplant. Alpha-SMA- and BMP4-positive cells were observed near the root surface at 5 days after transplantation. With the progress of alveolar bone regeneration, osteoblasts expressing Runx2 and Osterix appeared in the bone-forming region. These results indicate that periodontal ligament tissue remaining on the root surface after a tooth extraction contains undifferentiated cells that have the ability to regenerate alveolar bone. The process of osteoblast differentiation in this model might be similar to that for normal alveolar bone formation. Thus, periodontal ligament cells might be useful for the regeneration of alveolar bone in tissue engineering applications.

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.

Regeneration of alveolar bone is essential for periodontal treatment. Recently, cell replacement therapy has been focused on periodontal disease, but the source of the cells that regenerate alveolar bone is still uncertain. Therefore, to clarify the source of these bone-regenerating cells, we transplanted GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Five days after transplantation, the tooth was surrounded by connective tissue containing many blood vessels. At 10 days, bone-like tissue was formed in the connective tissue between the branches of the bifurcated root. This hard tissue expanded to almost all of this bifurcation area without osseous ankylosis after 20 days. All osteoblast-like cells in the newly formed matrix were immunopositive for GFP. In addition, these cells and the peripheral cells of the matrix showed intense immunoreactivity for BMP4, Runx2, BSP, and OPN. These results demonstrate that periodontal ligament tissue contains osteoprogenitor cells that have the ability to regenerate alveolar bone. Our model suggests that these regeneration processes might be similar to normal alveolar bone formation.

While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

Objectives GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. Materials and methods The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14 days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21 days. Results The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. Conclusion GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

The dental follicle contains mesenchymal cells that differentiate into osteoblasts, cementoblasts, and fibroblasts. However, the characteristics of these mesenchymal cells are still unknown. alpha-Smooth muscle actin (alpha-SMA) is known to localize in stem cells and precursor cells of various tissues. In the present study, to characterize the undifferentiated cells in the dental follicle, immunohistochemical localization of alpha-SMA was examined during rat molar tooth development. Rat mandibles were collected at embryonic days (E) 15-20 and postnatal days (P) 7-28. Immunohistochemical stainings for alpha-SMA, periostin, Runt-related transcription factor-2 (Runx2), tissue nonspecific alkaline phosphatase (TNAP), and bone sialoprotein (BSP) were carried out using paraffin-embedded sections. alpha-SMA localization was hardly detected in the bud and cap stages. At the early bell stage, alpha-SMA-positive cells were visible in the dental follicle around the cervical loop. At the late bell to early root formation stage (P14), these cells were detected throughout the dental follicle, but they were confined to the apical root area at P28. Double immunostaining for alpha-SMA and periostin demonstrated that alpha-SMA-positive cells localized to the outer side of periostin-positive area. Runx2-positive cells were visible in the alpha-SMA-positive region. TNAP-positive cells in the dental follicle localized nearer to alveolar bone than Runx2-positive cells. BSP was detected in osteoblasts as well as in alveolar bone matrix. These results demonstrate that alpha-SMA-positive cells localize on the alveolar bone side of the dental follicle and may play a role in alveolar bone formation.

To characterize osteodentin in the pre-functional rat incisor, we performed histological and histochemical evaluation of the anterior apex of the incisor in 3-day-old rats. The unerupted incisor was composed of osteodentin, with numerous cells present in the anterior apex. The osteodentin was immunopositive for osteocalcin, bone sialoprotein, and dentin sialoprotein, with an immunolocalization pattern similar to that of dentin. Back-scattered electron microanalysis (BSE) and electron probe microanalysis (EPMA) showed that osteodentin was not uniformly calcified. These results indicate that osteodentin in the rat incisor possesses dentin-like characteristics, and may be fragile in structure.

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.

The gene expression and protein distribution of matrix metalloproteinase (MMP) -2, -9, membrane type-1 MMP (MT1-MMP), as well as of TIMP-1, -2, and -3 were analyzed during mouse molar development. Immunohistochemical data demonstrated that all the MMPs investigated were expressed in the dental epithelium and mesenchyme. In contrast, gene and protein expression analysis for TIMPs showed that they were differentially expressed. TIMP-1 was expressed in the dental epithelium and mesenchyme between E13 and E16 and was transiently up-regulated at E14, the cap stage. TIMP-1 expression was also detected in differentiating odontoblasts. TIMP-2 RNA transcripts were found in the peridental and dental mesenchyme, odontoblasts, and ameloblasts. Protein analysis revealed high expression on the lingual side of the dental epithelium and underlying mesenchyme together with transient expression in the enamel knot at E14 and expression in the gingival tissue and enamel matrix postnatally. TIMP-3 RNA transcripts were found in discrete regions of the dental epithelium, including at high levels in the cervical loop at E16. Expression was also detected in preodontoblasts at E16 and transiently during ameloblast differentiation. Analysis of the protein distribution revealed a lower level of TIMP-3 on the lingual side of the dental epithelium at E14. MT1-MMP was expressed in the dental mesenchyme between E13 and E16, at relatively high levels in the cervical loop at E14, and in the odontoblasts and ameloblasts. The distinct temporospatial distribution patterns of the TIMPs suggest that these inhibitors play several intrinsic roles during tooth development. Developmental Dynamics 228:105–112, 2003. © 2003 Wiley-Liss, Inc.

Major histocompatibility complex (MHC) class II molecule-expressing cells are distributed in human dental pulp, and have been shown to accumulate beneath caries lesions. The responses of these cells and nerve fibers were analyzed under 5 different clinical conditions: shallow and deep experimental cavities, active and slow untreated caries, and treated caries. Under deep cavities, class II molecule-expressing dendritic cells displaced the injured odontoblasts during a period of one month, while such a response was not observed in shallow cavities and untreated or treated carious teeth. The class II molecules seen in the neural elements under active caries were no longer detectable in treated carious teeth. However, six months after treatment, clusters consisting of dendritic cells, T-lymphocytes, and nerve fibers still remained locally in the subodontoblastic area. These results indicate that dental pulps respond differently to cavity preparation and restoration between normal and caries conditions, and that immunoresponses persist for many months, even after caries treatment.

In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.

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.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).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.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.

Most targeted gene mutations are recessive and analyses of gene function often focus on homozygous mutant phenotypes. Here we describe parts of the expression pattern of M-twist in the head of developing wild-type mice and present our analysis of the phenotype of heterozygous twist- null animals at around birth and in adults. A number of twist -null heterozygous mice present skull and limb defects and, in addition, we observed other malformations, such as defects in middle ear formation and the xyphoïd process. Our study is of interest to understand bone formation and the role of M-twist during this process, as within the same animal growth of some bones can be accelerated while for others it can be delayed. Moreover, we show here that expressivity of the mouse mutant heterozygous phenotype is dependent on the genetic background. This information might also be helpful for clinicians, since molecular defects affecting one allele of the human H-twist ( TWIST ) gene were identified in patients affected with Saethre-Chotzen syndrome (SCS). Expressivity of this syndrome is variable, although most patients present craniofacial and limb malformations resembling those seen in mutant mice. Thus the mutant mouse twist -null strain might be a useful animal model for SCS. The twist -null mutant mouse model, combined with other mutant mouse strains, might also help in an understanding of the etiology of morphological abnormalities that appear in human patients affected by other syndromes.

Nerve fibers and class II major histocompatibility complex (MHC) antigen-expressing dendritic cells have been known to gather in the dental pulp beneath carious lesions. Significant functional interactions presumably occur between the neural and immune elements. The present study analyzed the morphological relationship between class II-expressing cells and nerve fibers in fuman carious teeth, visualized by a HLA-DR monoclonal antibody and a protein gene product 9.5 (PGP 9.5) polyclonal antibody; a confocal laser scanning microscope (CLSM) and an electron microscope were used. In pulps affected by early caries, HLA-DR-positive dendritic cells aggregated mainly in the cell-free zone associated with bundles of PGP 9.5-immuno-reactive nerve fibers. In pulps affected by advanced caries, the accumulated HLA-DR-positive cells and PGP 9.5-immunoreactive nerve fibers showed close association with each other especially beneath the odontoblast layer: the cells even embraced the nerve fibers. Intriguingly, class II molecules were recognized not only in dendritic cells but also in the Schwann cells of non-myelinated nerves in the pulp. Using immuno-electron microscopy, class II molecules were localized on the surface of the non-myelinating Schwann cells and also within some vesicles, whereas myelinating Schwann cells lacked this immunoreactivity. PGP 9.5-immunoreactive nerve fibers were also observed densely in the odontoblast layer, and CLSM revealed an intimate association of the nerve fibers and dendritic cells. The immunoreactivity for HLA-DR in Schwann cells depended upon the severity of the carious lesion. Class II-expressing Schwann cells are suggested to function as antigen-presenting cells in addition to dendritic cells.

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.

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 PGE 2 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 PGE 2 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 PGE 2 released from the LPS-inflamed pulp ( P 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 PGE 2 . The significant decrease in PGE 2 release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE 2 in the transmembrane efflux pathway.

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.

Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.

Dental pulp is involved in the formation of bone-like tissue in response to external stimuli. However, the origin of osteoblast-like cells constructing this tissue and the mechanism of their induction remain unknown. We therefore evaluated pulp mineralization induced by transplantation of a green fluorescent protein (GFP)–labeled tooth into a GFP-negative hypodermis of host rats. Five days after the transplantation, the upper pulp cavity became necrotic; however, cell-rich hard tissue was observed adjacent to dentin at the root apex. At 10 days, woven bone-like tissue was formed apart from the dentin in the upper pulp. After 20 days, these hard tissues expanded and became histologically similar to bone. GFP immunoreactivity was detected in the hard tissue-forming cells within the root apex as well as in the upper pulp. Furthermore, immunohistochemical observation of α–smooth muscle actin, a marker for undifferentiated cells, showed a positive reaction in cells surrounding this bone-like tissue within the upper pulp but not in those within the root apex. Immunoreactivities of Smad4, Runx2, and Osterix were detected in the hard tissue-forming cells within both areas. These results collectively suggest that the dental pulp contains various types of osteoblast progenitors and that these cells might thus induce bone-like tissue in severely injured pulp.

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.

Introduction The degradation of fibrillins, the major constituents of microfibrils, is known to facilitate the release of active transforming growth factor-β (TGF-β), 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-β-binding protein (LTBP)-1, matrix metalloproteinase-3 (MMP-3), alkaline phosphatase (ALP), and in situ hybridization of fibrillin-1. Pulp tissue slices cultured with β-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 β-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 β-glycerophosphate–induced pulpal mineralization in vitro .

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.

This paper aims to review the biological and physicochemical properties of mineral trioxide aggregate (MTA) with respect to its ability to induce reparative dentinogenesis, which involves complex cellular and molecular events leading to hard-tissue repair by newly differentiated odontoblast-like cells. Compared with that of calcium hydroxide-based materials, MTA is more efficient at inducing reparative dentinogenesis in vivo. The available literature suggests that the action of MTA is attributable to the natural wound healing process of exposed pulps, although MTA can stimulate hard-tissue-forming cells to induce matrix formation and mineralization in vitro. Physicochemical analyses have revealed that MTA not only acts as a “calcium hydroxide-releasing” material, but also interacts with phosphate-containing fluids to form apatite precipitates. MTA also shows better sealing ability and structural stability, but less potent antimicrobial activity compared with that of calcium hydroxide. The clinical outcome of direct pulp capping and pulpotomy with MTA appears quite favorable, although the number of controled prospective studies is still limited. Attempts are being conducted to improve the properties of MTA by the addition of setting accelerators and the development of new calcium silicate-based materials.