Masaaki Iwaku

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 postoperatively. At 14 days, an alignment of cells, some of which were elongated and odontoblast-like, was observed adjacent to the fibroriectin-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.

Bacteria Invading Periapical Cementum

The aim of this study was to determine whether microorganisms invade periapical cementum of human teeth from the adjacent periapical lesions. We therefore attempted to isolate microorganisms from periapical cementum through the adoption of standard anaerobic procedures for obligate anaerobes. Samples of cementum were taken from 10 amputated tooth roots at the time of apicoectomy. From two of these samples, bacteria were recovered after anaerobic incubation, but no bacteria were recovered after aerobic incubation of the same samples. Of a total of eight isolates from the cementum, seven were obligate anaerobes and one was aerotolerant. The obligate anaerobes isolated were assigned to the genera Prevotella, Peptostreptococcus, Eubacterium, and Fusobacterium. The aerotolerant anaerobe was Campylobacter. From this, we conclude that bacteria can successfully invade cementum via periapical periodontal tissue, and that such bacteria may play a significant role in chronic periapical pathosis.

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 Hertwigs 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.

Bactericidal Efficacy of Metronidazole against Bacteria of Human Carious Dentin in vitro

The bactericidal efficacy of metronidazole against bacteria in carious dentin was clarified by measuring (1) the difference between bacterial recovery from suspensions of carious dentin on metronidazole-containing BHI-Blood agar plates (10 micrograms/ml) and control plates and (2) the difference between bacterial recovery from carious lesions of freshly extracted teeth, covered by alpha-tricalcium phosphate (TCP) cement containing metronidazole (5%) for 1-3 days and that covered by TCP only. More than 10(3) bacteria per milligram sample were recovered from carious dentin. More than 99% of the bacteria were, however, not recovered when samples were inoculated on metronidazole-containing BHI-Blood agar plates or when the lesions were covered by TCP cement containing metronidazole, indicating that metronidazole effectively disinfected the carious dentin.

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

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

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.

A Confocal Laser Scanning Microscopic Study of The Immunofluorescent Localization of Fibronectin in The Odontoblast Layer of Human Teeth

The distribution of fibronectin in dental pulp was studied in developing and developed human teeth by indirect immunofluorescence using a confocal laser scanning microscope. In the apical region of developing teeth, intense fluorescence was found along the basement membrane facing the mesenchyme of Hertwigs epithelial sheath and first-formed (mantle) predentine. With further elongation of odontoblasts, fibronectin was observed between the cells, appearing as corkscrew fibres passing from the pulp into predentine parallel to the long axis of the odontoblasts. In the coronal region of developing and developed teeth a similar distribution of fibronectin was observed in the odontoblast layer. At the border zone between odontoblasts and predentine the reaction was intense, but was weak in the predentine itself. In the calcified dentinal matrix it had disappeared completely, except for the area along the dentinal tubules. The results demonstrate that fibronectin is present in the odontoblast layer during all stages of dentinogenesis. Fibronectin-positive fibrous structures between odontoblasts probably correspond to von Korff fibres, and are closely related to odontoblast differentiation and dentinogenesis.

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.

The metabolism of phenylalanine and leucine by a cell suspension of Eubacterium brachy and the effects of metronidazole on metabolism

Degradation of phenylalanine and leucine by resting cells of Eubacterium brachy ATCC 33089 were studied under strict anaerobic conditions. The effects of metronidazole and air on the metabolism were also studied. The organism principally produced phenylpropionate and isocaproate from phenylalanine and leucine, respectively. Other products were cinnamate and phenylacetate from phenylalanine, and alpha-ketoisocaproate and isovalerate from leucine. The organism also produced hydroxylated end-products, i.e. phenyllactate from phenylalanine and hydroxyisocaproate from leucine. When metronidazole was added to the reaction mixture, the production of phenylpropionate, cinnamate, phenylacetate, isocaproate, alpha-ketoisocaproate and isovalerate was inhibited, while that of hydroxylated products was not, suggesting that the organism has metronidazole-sensitive and -tolerant pathways of metabolism. A similar inhibitory effect was also found when the reaction was done aerobically, suggesting that the inhibitory mode of metronidazole is similar to that of oxygen.