Gli1+-PDL Cells Contribute to Alveolar Bone Homeostasis and Regeneration.

The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1+ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1+ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1+ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.

M2 Phenotype Macrophages Colocalize with Schwann Cells in Human Dental Pulp.

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

Detection of bone marrow-derived fibrocytes in human dental pulp repair.

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.

Bioactivity and biomineralization ability of calcium silicate-based pulp-capping materials after subcutaneous implantation.

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 analysed with scanning electron microscopy-electron probe microanalysis (SEM-EPMA). The distributions of calcium (Ca) and phosphorus (P) at the material-tissue interface were also analysed with SEM-EPMA. Comparisons of the thicknesses of the Ca- and P-rich areas were performed using the 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 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). CONCLUSION: 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.

GaAlAs laser-induced pulp mineralization involves dentin matrix protein 1 and osteopontin expression.

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.

Temporospatial localization of dentine matrix protein 1 following direct pulp capping with calcium hydroxide in rat molars.

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 < 0.05) and then decreased. CONCLUSIONS: The deposition of DMP1 at exposed pulp sites preceded the appearance of nestin-immunoreactive cells, active cell proliferation and new matrix formation after pulp capping with calcium hydroxide in rat molars, suggesting that DMP1 acts as a trigger of pulp repair. The colocalization of DMP1 and osteopontin suggests that these two proteins play complementary roles.

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

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

Hard tissue formation in subcutaneously transplanted rat dental pulp.

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.

Class II antigen-presenting dendritic cell and nerve fiber responses to cavities, caries, or caries treatment in human teeth.

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.

Differential expression of laminin-5 subunits during incisor and molar development in the mouse.

Rodent incisors are continuously growing teeth and enamel deposition is restricted to the labial side. In the present study, the expression of laminin-5 subunits (alpha3, beta3 and gamma2) has been analyzed by in situ hybridization in developing mouse lower incisors and compared to that reported in the molar. At the bud stage (E12), mRNAs for all subunits were detected in the whole epithelial thickening. At E14, when histogenesis had started, transcripts for alpha3 and gamma2 subunits were restricted to the outer dental epithelium (ODE), whereas the beta3 subunit was intensely expressed in the inner dental epithelium (IDE). A transient expression for alpha3 subunit was seen in the enamel knot area and disappeared at E15. Subsequently, all laminin-5 subunit genes were re-expressed in differentiating ameloblasts on the labial side. Similar patterns of transcription were observed in incisor and molar, suggesting that the differential expression of laminin-5 subunits in the IDE might be involved in the histogenesis of the IDE and ameloblast differentiation. At E16.5, cells of the IDE at the anterior extremity of the incisor and in the anterior part of the lingual IDE expressed transcripts for alpha3 and beta3 but not for gamma2 subunit. Similar expression patterns were observed in the enamel-free areas of the E18 molar. This specific expression might thus be related to cells that do not differentiate as functional ameloblasts. Throughout incisor development, intense expression for all laminin-5 subunits was restricted to the labial side of the cervical loop. The asymmetrical expression of laminin-5 might be related to incisor morphogenesis and to the differences in histogenesis and cytodifferentiation of the IDE that exist in the labial versus lingual aspect of the cervical loop.

Immunohistochemical localizations of class II antigens and nerve fibers in human carious teeth: HLA-DR immunoreactivity in Schwann cells.

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.

Expression and localization of laminin-5 subunits in the mouse incisor.

Laminin-5 is associated with several epithelial tissues and forms part of the anchoring filaments of hemidesmosomes. Recent data have shown that the expression of laminin-5 subunits is impaired in junctional epidermolysis bullosa (JEB), and, in these patients, enamel hypoplasia is commonly observed. Rodent incisors are continuously growing teeth with an asymmetry between their labial and lingual sides. Enamel matrix formation is restricted to the labial side. We have analyzed the changes in the expression and localization of laminin-5 subunits (alpha3, beta3, and gamma2) in lower incisors of the mouse. The apical loop located at the end of the labial side contained stem cells and showed expression for all laminin-5 subunits. In the anterior direction, the inner dental epithelial cells (IDE) transiently lost the immunoreactivity for all subunits, whereas the transcripts for the beta3 subunit remained in the IDE. All subunit mRNAs and proteins were expressed in ameloblasts facing predentine and also in secretory and maturation stage ameloblasts. Enamel matrix contained laminin-5. On the lingual side, the expression of laminin-5 subunits was continuous from the epithelial root sheath to the epithelial rests of Malassez in the periodontal ligament. These results suggest that spatial and temporal regulation of laminin-5 subunits correlates with the histogenesis of the dental organ, ameloblast differentiation, and enamel formation and also that laminin-5 plays a role in the adhesion between dental epithelial cells and the extracellular matrix (enamel or dentine) in areas where the dental basement membrane is absent.

Expression and localization of laminin-5 subunits during mouse tooth development.

Tooth morphogenesis is regulated by epithelial-mesenchymal interactions mediated by the basement membrane (BM). Laminins are major glycoprotein components of the BMs, which are involved in several cellular activities. The expression and localization of the alpha3, beta3, and gamma2 laminin-5 subunits have been analyzed by in situ hybridization and immunohistochemistry during mouse molar development. Initially (E12), mRNAs of all subunits were detected in the entire dental epithelium and the corresponding proteins were located in the BM. During cap formation (E13-14), transcripts for the alpha3 and gamma2 subunits were localized in the outer dental epithelium (ODE), whereas the beta3 subunit mRNA was present in the inner dental epithelium (IDE). During the early bell stage (E16), immunoreactivity for all subunits disappeared from the BM along the IDE, although intense signals for beta3 mRNA were detectable in cells of the IDE. Subsequently, when the dentinal matrix was secreted by odontoblasts (E18-19.5), mRNAs of all three subunits were re-expressed by ameloblasts, and the corresponding proteins were detected in ameloblasts and in the enamel matrix. Tissue recombination experiments demonstrated that when E16 IDE or ODE was associated with E18 dental papilla mesenchyme, immunostaining for all laminin-5 subunits disappeared from the BM, whereas when cultured with non-dental limb bud mesenchyme, they remained positive after 48 hr of culture. These results suggest that the temporospatial expression of laminin-5 subunits in tooth development, which appears to be differentially controlled by the dental mesenchyme, might be related to the enamel organ histo-morphogenesis and the ameloblast differentiation.

Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program.

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.

Immunolocalization of fibronectin during reparative dentinogenesis in human teeth after pulp capping with calcium hydroxide.

Exposed dental pulp is known to possess the ability to form a hard-tissue barrier (dentin bridge). The exact mechanisms by which pulp cells differentiate into odontoblasts in this process are unknown. Fibronectin has been demonstrated to play a crucial role in odontoblast differentiation during tooth development. This study tested the hypothesis that fibronectin is involved in the initial stages of replacement odontoblast differentiation and reparative dentin formation. We observed its immunohistochemical localization during dentin bridge formation in human teeth, after pulp was capped with calcium hydroxide [Ca(OH)2]. One day after the capping, precipitation of crystalline structures was observed at the TEM level in association with cell debris at the interface between the superficial necrotic zone and underlying pulp tissue. This layer of dystrophic calcification showed positive reaction for fibronectin, and pulp cells appeared to be closely associated with this layer, seven to ten days post-operatively. At 14 days, an alignment of cells, some of which were elongated and odontoblast-like, was observed adjacent to the fibronectin-positive irregular matrix. Between the cells, corkscrew fiber-like fluorescence was visible. At 28 days, the irregular fibrous matrix was followed by the formation of tubular dentin-like matrix lined with odontoblast-like cells. Therefore, it would seem that fibronectin associated with the initially formed calcified layer might play a mediating role in the differentiation of pulp cells into odontoblasts during reparative dentinogenesis, after pulp was capped with Ca(OH)2.

Immunohistochemical localization of HLA-DR-positive cells in unerupted and erupted normal and carious human teeth.

Class II major histocompatibility complex (MHC) antigen-expressing cells are generally associated with the early phase of the immune response. We have studied the distribution of class II-expressing cells in developing, normal, and carious human teeth to clarify when human pulp acquires an immunologic defense potential and how this reacts to dental caries. Antigen-expressing cells were identified immunohistochemically by means of HLA-DR monoclonal antibody. In the pulp of unerupted developing teeth, numerous HLA-DR-positive cells were distributed mainly in and around the odontoblast layer. In erupted teeth, HLA-DR-positive cells were located, for the most part, just beneath the odontoblast layer, with slender cytoplasmic processes extending into the layer. Superficial caries lesions caused an aggregation of HLA-DR-positive cells in dental pulp corresponding to the lesion. In teeth with deeper caries lesions, this aggregation of cells expanded to include the odontoblast layer. Also noted were HLA-DR-positive cells lying along the pulp-dentin border, with cytoplasmic processes projecting deep into the dentinal tubules, where they co-localized with odontoblast processes. These findings suggest that: (1) human dental pulp is equipped with immunologic defense potential prior to eruption; (2) in the initial stage of caries infection, an immunoresponse mediated by class-II-expressing cells is initiated in human dental pulp; and (3) HLA-DR-positive cells trespass deep into dentinal tubules as the caries lesion advances.

Immunolocalization of the small proteoglycan decorin in human teeth.

The immunolocalization of decorin was studied by confocal laser scanning microscopy and transmission electron microscopy. In the apical area of developing teeth, labelling for decorin was found in the dental papilla cells, prodontoblasts and also in the Hertwig's epithelial cells. Mantle dentine and the initial predentine were negative. In circumpulpal dentine, intense reactivity extended along the calcification front and dentinal tubules. Fluorescence was also evident in odontoblast cell bodies and their processes in predentine. None was perceived, however, in the predentinal matrix. Faint staining was observed on the calcified dentinal matrix. Immunoelectron microscopy revealed staining for decorin in collagen fibrils lining the predentine-dentine junction, and where arrays of labelled filaments were noted orthogonal to the collagen fibrils. Staining extending from the calcification front was observed in the matrix adjacent to the dentinal tubule. The decorin observed at the calcification front might regulate the mineralization of dentinal matrix.

Immunoelectron-microscopic study of the localization of fibronectin in the odontoblast layer of human teeth.

Indirect immunofluorescence-based studies have shown similarities in the distribution patterns of fibronectin-positive fibrous structures and so-called von Korff fibres. The aim of the present study was to analyse the reactivity of fibronectin in the odontoblast layer of fully developed human teeth by means of immunoelectron microscopy. Between the odontoblasts, discrete and undulatory fibrillar fascicles with peroxidase labelling were observed. They seemed to be in contact with odontoblasts in some areas, while in others they appeared to be intervening between two neighbouring odontoblasts. Higher magnifications of the fibrillar material demonstrated axial periodic staining of about 70 nm. Peroxidase reaction of fibronectin was also recognized along the cell membrane of odontoblasts facing predentine. The fibronectin in fibrillar fascicles observed between odontoblasts would be held in place by the direct molecular interaction with collagen fibrils and contribute to the pulpward migration of these cells and maintenance of their specific morphology. At the distal end of odontoblasts, a tight seal would be maintained by means of odontoblast-fibronectin adhesion.

Effects of antibacterial capping agents on dental pulps of monkeys mechanically exposed to oral microflora.

The effects of antibacterial drugs on bacterially contaminated dental pulps were investigated in monkeys. Class V buccal cavities with pulpal exposures were prepared and then left open to the oral environment for 24 h. The exposed pulps were capped with alpha-tricalcium phosphate (alpha-TCP) containing a mixture of antibacterial drugs. Either alpha-TCP or Ca(OH)2 was used as a control. Pulpal responses were histologically evaluated after 4 wk. Those teeth capped with alpha-TCP alone showed total pulp necrosis and bacterial growth within the pulp chamber. By contrast, the pulps capped with alpha-TCP containing mixed antibacterial drugs remained almost normal without any necrotic layer, but showed persistent absorbing response to capping materials and no signs of hard tissue barrier formation. In teeth capped with Ca(OH)2, a hard tissue barrier was formed below the exposure site, with a wide loss of pulp tissue. No inflammation was seen under the barrier. These results indicate that mixed antibacterial drugs added to alpha-TCP effectively disinfected pulpal lesions, without destroying any of the sound pulp tissue. However, hard tissue barrier formation was delayed by this mixture as compared with Ca(OH)2.

Histological observations of hard tissue barrier formation in amputated dental pulp capped with alpha-tricalcium phosphate containing calcium hydroxide.

This study was performed to evaluate the pulpal response to alpha-tricalcium phosphate (alpha TCP) containing calcium hydroxide (Ca(OH)2). The dental pulps of monkeys were amputated and dressed with four agents: alpha TCP, alpha TCP containing 1% Ca(OH)2, alpha TCP containing 5% Ca(OH)2, and Ca(OH)2 being used as a control. The pulpal responses were histologically evaluated after 4 and 8 weeks. The pulp tissue treated with alpha TCP proliferated above the level of the original wound surface, and a thin layer of hard tissue barrier was formed directly against the capping agent. The barrier demonstrated atubular matrix lined with flattened or cuboidal cells, but occasionally appeared irregular in form. Ca(OH)2 dressing resulted in destruction of pulp tissue, with a thick hard tissue barrier being formed below the level of the exposure site. The barrier consisted coronally of osteodentin and pulpally of tubular dentin lined with odontoblast-like cells. By contrast, 1% Ca(OH)2 added to alpha TCP produced a slight proliferation of pulp tissue. An atubular matrix barrier, pulpally lined with cuboidal cells, formed above the exposure site. It was later followed by the formation of tubular matrix lined with columnar cells. Teeth treated with 5% Ca(OH)2 showed a thin necrotic layer and a thick barrier formation. The barrier was composed of tubular dentin-like tissue lined with odontoblast-like cells. It would appear that alpha TCP containing a small amount of Ca(OH)2 may be clinically useful as a capping agent, as it induced consistent hard tissue formation, without excessive destruction of underlying pulp tissue.