Dentinal Fibers Research Articles

The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis

ObjectiveThe bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix. DesignWe generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts. ResultsThe first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype. ConclusionsThese data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth.

Effect of Er:YAG laser energy densities on thermally affected dentin layer: Morphological study.

Physical and chemical composition of dentin is subject to modification when irradiated with Er:YAG laser. Temperature rise causes water evaporation and micro-mechanical ablation of dentin. The misuse of laser parameters could affect negatively dentin collagen fibers leading to failure in bonded composite restorations. The aim of this in vitro study was to evaluate the effect of Er:YAG laser radiation at different levels of energy on the morphology of thermally affected dentin layer. Forty-eight freshly extracted human third molars were randomly divided into six groups (n = 8). In all groups, except for the control groups, dentin was subject to irradiation with H02 handpiece Er:YAG laser in non-contact mode (SSP mode = 50 µs; 10 Hz; speed of 1 mm/second; air 6 mL/min; and water 4 mL/min) with the following levels of energy (40, 60, 80, 100, and 120 mJ) respectively. Teeth were sliced longitudinally. Photo-ablated cavities were observed. The cavity depth and dentin fiber collagen deterioration were measured. Laser irradiation increased the depth of dentinal crater from 46.57 µm to 178.2 µm, when energy level increased from 40 mJ to 120 mJ. A superficial black layer, representing dentinal affected collagen fibers, was present in all groups except for control group. When comparing the thickness of the black layer, there was no significant difference between groups. It increased at 40 mJ to 28.17 µm then decreased to 15.19 µm at 60 mJ and then increased again for 80 mJ to 19.93 µm, 100 mJ to 22.87 µm and 120 mJ to 28.53 µm. Only one group (60 mJ) showed low values and significant difference as compared to the other irradiated groups, when multiple comparisons tests (ANOVA) were made using Newman-Keuls test. Dentin organic matrix presented the minimum alteration when Er:YAG laser is used specifically at an appropriate level of energy (60 mJ).

Characterization of Enamelysin (MMP-20) Expressed in the Porcine Odontoblast

Enamelysin (MMP-20) is a matrix metalloproteinase isolated from the cDNA library of the enamel organ. Although enamelysin is also expressed in the tooth pulp, especially in the odontoblast, the functional significance of enamelysin protein in dentinogenesis has not been elucidated. Thus, the present study was designed to clarify the characteristics of enamelysin in the pulp from the porcine deciduous molar tooth germ and developing teeth. Western blot analysis of proteins extracted from the pulp of the tooth germ revealed that immunoreactive enamelysin occurred as 54 kDa and 51 kDa proteins. None of the protein bands lower than 51 kDa was stained. Light and electron microscopic immunohistochemistry of tooth germs and erupting teeth demonstrated positive reactions over the cytoplasmic matrix of odontoblast cell bodies and processes (dentinal fibers). The secretory machinery of the odontoblast was shown to be devoid of immunoreactivity. Intensity of the immunoreaction in the odontoblast was variable but essentially dependent upon the position and differentiation stage of the odontoblast. The highest immunoreactivity was found in some odontoblasts located at the cuspal region of the tooth germ. During tooth eruption, cell bodies of odontoblasts in the crown showed faint or no immunoreactivity. No immunoreaction was detected in the odontoblast facing the root dentin, although dentinal fibers of the root dentin showed faint immunoreactivity. RT-PCR and cDNA sequence analysis failed to find any differences between enamelysin mRNA extracted from the enamel organ and that from the pulp. These results suggest that odontoblasts transiently express enamelysin during tooth formation and that the enamelysin in the odontoblast is not secreted from the cell.

支台歯形成後の歯髄変化に関する光顕ならびに電顕的研究

This study was aimed to investigate histo-pathological changes in the pulp of the dog incisors (240 teeth) after full crown preparation by light, transmission and scanning electron microscopy. The results obtained were summarized as follows : 1. Scanning electron microscopy revealed that dentinal tubules visualized by full crown preparation always contained torn dentinal fibers in the cutting surface of the dentin. This finding suggested that the initial changes of the pulp were caused by the direct damage to odontoblasts. 2. Changes caused by the preparation occurred widely in the coronal pulp, particularly in the pulp horn regions. These changes were dependent on the thickness of remaining dentin. 3. The recovery of odontoblast layer was observed at 3 days after preparation : Undifferentiated pulp cells were seen increasing their number at the site just beneath the odontoblast layer. Undifferentiated pulp cells located just beneath the odontoblast layer were observed to make contact with each other by gap junctions. These peculiar contacts were also observed frequently between this type of cells and odontoblasts. Further, similar cells were also found among injured odontoblast. It was suggested from the present morphological view points that these undifferentiated cells were precursor cells of odontoblasts and migrated into the odontoblast layer. 4. The reparative dentin began to form 7 days after preparation and increased its thickness gradually. The thickness of the reparative dentin was estimated to be about 40μm at the lingual or buccal side of pulp at 28 days after operation. 5. Matrix vesicle-like structures were seen in the predentin where newly formed odontoblasts aggregated. 6. The pathological changes in the pulp almost disappeared by 21 days after preparation. 7. Exposed dentinal fibers were still observed on the cutting surface even 28 days after preparation by scanning electron microscopy. Possibility raised from the present study that the pulp was still influenced by the oral environment through dentinal fibers even long period after preparation. For getting a good prognosis, the adequate post-treatments, that is, use of temporary crowns and suitable cements, seemed to be necessary.

切削荷重が歯髄へ及ぼす影響

In order to investigate the effect of the difference in magnitude of cutting load, one of the cutting conditions, on the dental pulp immediately after cutting in cavity preparation by high-speed cutting on vital tooth, a cutting device capable of exerting a given cutting load was designed. With the use of this device, 80g or 120g cutting load was applied on the six lower anterior teeth of dogs in performing cutting preparation, and light and electron microscopic observations were made. The results were as follows : 1. Scanning electron microscopic observation of the three dimensional structure of dentinal fibers of non-cut dogs' teeth with application of cryofracture and the HCl-collagen digestive method showed that most of the dentinal fibers, as in the human teeth, ran in the three dimensional curve from the odontoblasts arranged in a line and reached the enamel-dentin junction. 2. Light microscopic observation of cavity preparated teeth of dogs showed irregular arrangements, reticular atrophy, and appearance of aspirated bodies in the odontoblasts under the cavity in both 80g and 120g cutting load cases. At the same time, no significant changes were observed in the proper dental pulp under the odontoblasts. 3. Aspirated bodies appeared in 52% of cavity preparations with 80g cutting load and in 60% of 120g load. 4. The fact that the aspirated bodies which had appeared immediately after cavity preparation showed Feulgen positive nuclear reaction suggested that each was the nucleus of the odontoblast. 5. Transmission electron microscopic observation showed morphological changes in odontoblasts in both 80g and 120g preparation cases. Namely, atrophy, pyknosis, and aspiration of some or most the nuclei of odontoblasts which penetrate into the dentinal tubules were observed. However, alterations of the odontoblasts in 120g preparation were more conspicuous than those of 80g preparation. Almost all of the aspirated bodies observed in 120g preparation were located deeply in the dentinal tubules and their nuclei occasionally showed dissolutive appearance. 6. It was suggested that one of the pathogenesis for the appearance of aspirated bodies might result from dragging-in of the dentinal fibers by air-turbine revolution. 7. It was observed in this study that the less cutting load made the impairment to the odontoblast slighter. The necessity of less cutting load in clinical work was confirmed again.

Electron microscopy of cells displaced into the dentinal tubules due to dry cavity preparation.

Using dogs' teeth, cells displaced into the dentinal tubules due to dry cavity preparation were observed using electron microscopy. In order to obtain adequate fixation a new local-perfusion method was employed. Most of the cells which moved into the dentinal tubules were odontoblast nuclei and partly neutrophilic leukocytes and erythrocytes. Not only odontoblast nuclei but also cytoplasmic organelles, such as mitochondria, rough endoplasmic reticulum, free ribosomes, and lysosomes were displaced into the tubules. The weavings of dentinal fibers in the tubules were observed in some areas.

健常家兎象牙質ならびに切削によるその形態変化に関する電子顕微鏡的観察

Recently, many contributions has been given on the problem of the teeth grindings. The present study which applied to the rabbit was the electron microscopic observations to clear up ultrafine-structures of the normal dentin and morphological changes of the dentin occurred by using high speed dental engine for cavity preparation through the technique of undecalcified sectioning from the viewpoint of ultramicrophase. This observations for the normal dentin were conducted on the region just over labial alveolar crest of the incisors, as shown diagram, and for the experimental dentin, on the cases of three to twenty-four days after cavity preparation in the same region under the condition of the minimum irritation. The results were summarized as follows. On the normal dentin In the young dentin layer, the minimum diameter of dentinal tubules was approximately two microns and there were found within the tubules the odontoblastic process with network structures and vesicles, which were limited with delicate unit membrane. An inorganic fine granules accumulating on the typical collagen fibrils to some extent were observed in the matrix of this dentin layer. In the middle layer of calcified dentin, the minimum diameter of the tubules was about 1.5 microns. The dentinal fibers appeared to be surrounded by obscure limited membrane, and cytoplasm of the fibers changed into amorphous appearance different from the young layer. The characteristic periodicity of the collagen fibrils could not to be recognized completely because of fine granules accumulation in large quantities, and then obvious distinction between peritubular and intertubular matrices was shown. In the outward layer of the dentin, the fine containings within the tubules were found to be gathered in center or wall side of the tubules. The wall of the tubules showed various irregular appearances. The whole of the matrix appeared to be calcified similarly so that could not to be distinguished peritubular and intertubular substances. In the superfacial layer of the dentin, part of the terminal tips of the tubules could not to be recognized as they were filled with the hypercalcified matrix. On the experimental dentin Observations on the experimental dentin were undertaken on each cases of three days to twenty-four days at intervals of three days after the cavity preparation. There was not observed any changes in fine structure in young dentin of three and six days cases after the examination, that is, they were much the same as the findings of both of the tubules and the matrix in the normal dentin. During the duration of nine to fifteen days after the examination, variation in structure of the containings of the tubules were found, while the typical periodicity of the collagen fibrils was found obviously in part of the matrix. Middle layer of the dentin was demonstrated that the containings of the tubules was not only reduced, but also disappeared during the duration eighteen to twenty-one days after the examination. The matrix of the layer showed also reduce of the degree of calcification, since the typical periodicity of the collagen fibrils was found still obviously within limited region, whereby it was hardly possible to be distinguished peritubular and intertubular matrices. The outerward layer of the dentin underlying to the cavity floor did not indicate any clear changes. The dentinal fibers and matrix in the cases extended to twenty-four days after the operation was much the same structures as findings of those of the normal dentin. From the above findings, it was revealed that the reaction when the dentin was grinded by high speed dental engine under the condition of the least irritation, was not soon appeared, demineralization as related to the variations of the dentinal fibers occurred in the matrix after a certain period, and at twenty-four days after the operation the dentin was recovered completely from abnormal condition.

A pathological study on dental caries with labeled antibody techniques

The hypothesis that immunologic mechanism may play an important role in modifying the carious process was suggested by Blechman and Mori (1965). Presently an attempt was made to find the localization of salivary components, immunoglobulins, serum albumin and oral microorganisms in the human sound and carious dentin with a fluorescent antibody technique (Coons and Kaplan, 1950), an enzyme (peroxidase-labeled) antibody technique (Nakane and Pierce, 1967) and other staining techniques, as well as by microradiography.Seventy freshly extracted sound and carious human permanent teeth (Table 1) were immediately put in cold 95% ethanol for fixation. These were divided into 2 segments. A segment from each tooth was hand polished to a thickness of about 70μ, cooled with cold saline and used for microradiographic studies.Then it was demineralized in 0.5M pH7.4 EDTA (2Na-salt) at 4°C for 2-3 days. Another segment was demineralized in the same solution at 4°C for 7-14 days and embedded with paraffin. Microtome sections were made from each segment to a thickness of 7μ(Table 2).Rabbit antiserums to human saliva, human immunoglobulins*, human serum albumin* and oral microorganisms were prepared. Fluorescein isothiocyanate**, tetramethylrhodamine isothiocyanate** and horseradish peroxidase*** were conjugated with the globulin fraction of rabbit antiserum. Commercially available fluoresceinlabeled anti human immunoglobulins serum** was also used. Control studies of specificity were made with labeled antiserum from which specific antibody had been absorbed by the addition of antigen.When stained with labeled anti human saliva serum, labeled anti human immunoglobulins serum and labeled anti human immunoglobulins serum and labeled anti human serum albumin serum, specific reaction was usually observed in dentinal tubules (Figs. 6 and 7), dentinal fibers, predentin and the odontoblast layer of dentin (Fig. 8). These labeled antiserums also resulted in the most intense reaction in dentinal tubules beneath a translucent zone in the ground section, a radiopaque zone in microradiogram, of carious dentin (Figs. 10, 11, 14, 16, 17 and 18). Azure A eosin B stain, PAS stain and bromphenol blue stain produced a staining similar to that given by these labeled antiserums (Figs. 14, 15 and 19). Moreover, labeled anti human saliva serum resulted in diffuse reaction in soft carious dentin (Figs. 9 and 19). Although microorganisms were localized in dentinal tubules and the matrix of carious dentin (Figs. 20 and 21), no specific relation was observed between the localization of salivary components and that of oral microorganisms.*Nutritional Biochemicals Co., Cleveland, Ohio, U. S. A.**Baltimore Biological Laboratory, Baltimore, Maryland, U. S. A.***Sigma Chemical Co., St. Louis, Missouri, U. S. A.

Outgrowth of dentinal fibers observed in tissue culture

In hanging-drop cultures of dental pulp of the permanent canines and molars of newborn kittens, outgrowth of dentinal fibers from the outer surface of young dentin was observed when the enamel organ was removed. The outgrowth started soon after explantation and the fibers reached a considerable length in a short period of time. Outgrowing fibers were not ramified, but usually showed a knot near their distal end. The potency of growth at the peripheral end of the dentinal fibers was positively established.

A report of a case of hypoplasia of the dentine characterized largely by the relative failure in the formation of the dentinal matrix

A case of hypoplasia of the dentine was observed in an upper right second molar which was extracted from a patient over 40 years of age. The case was only accidentally observed in the course of our studies on the pulps of pyorrhetic teeth. Grossly, no abnormal condition was noted on the specimen before processing. The soft condition of the dentine was noted only after decalcification. Histologically, the main feature of the abnormal condition was the relative absence of dentinal matrix. The dentinal fibers or the fibers of Tomes were very poorly supported by ground substance of the dentine. Evidence of hypocalcification had been noted also in some areas of the tooth. The findings of the present case are different from the conditions observed in opalescent dentine (dentinogenesis imperfecta). To a certain extent, it resembles the reported case of “odontogenesis imperfecta” associated with osteogenesis imperfecta. The etiology of the case has not been determined. No detailed history and physical examination of the patient were made as we were not aware that there was something radically wrong or abnormal in the patient.