Calbindin D28K-like immunoreactive nerve fibres in the predentine of rat molar teeth

Immunoelectron-microscopy was applied to reveal the existence of nerve fibres and terminals showing calbindin D28k (CB)-like immunoreactivity (IR) in the rat molar tooth pulp. In the root pulp, thick, smooth-surfaced CB-IR nerve fibres were in bundles accompanying the blood vessels. In the coronal pulp, the fibres arborized repeatedly and extensively. CB-IR nerve fibres had a predominantly thick, smooth-surfaced appearance, though parts appeared thin and beaded. Occasionally some thin, varicose CB-IR nerve fibres ran along the odontoblasts, penetrating into the predentine alongside the dentinal tubules. They could be traced for approx. 10-20 microns into the predentine from the pulp-predentine border. Immunoelectron-microscopy revealed that only some of the nerve terminals in the predentine showed CB-IR, and that predentinal CB-IR nerve terminals were located close to the odontoblast processes. No synaptic structures were observed between them. The presence of CB-IR nerve terminals in the predentine suggests that many, if not all, CB-IR nerve fibres could be nociceptors. The CB could be involved in Ca2+ homeostasis during the activation of nociceptors.     

An ultrastructural study of the relationship between sensory trigeminal nerves and odontoblasts in rat dentin/pulp as demonstrated by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP)

Because the ultrastructure of the trigeminal sensory nerves in dentin, especially in relation to odontoblasts, remains to be clarified, we investigated the relationship between the trigeminal sensory nerves and the odontoblast processes using the anterograde axonal transport technique by injecting wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the rat trigeminal ganglion. Light microscopically, the nerves labeled with WGA-HRP were mainly concentrated at the pulpal horn, forming a nerve plexus at the subodontoblastic region and penetrating the predentin/dentin about 50 to 70 microns. Ultrastructurally, HRP reaction products were observed intra-axonally in the myelinated (A delta) and unmyelinated (C) axons in the subodontoblastic region. Most nerves lost the Schwann sheath and were naked in the predentin/dentin. The labeled varicosities were close to the odontoblast processes in the dentinal tubules. No synaptic structures could be detected between the varicosities and the odontoblasts, but a gap about 20 nm wide was found between them. One type of varicosity was a rich mitochondria-containing varicosity, while the other was a rich vesicle-containing (large dense core vesicles and small clear vesicles) one. The reaction products were also found in the extracellular spaces surrounding the axons. Sometimes the reaction products were seen in the coated pits or the endocytotic vesicles of the odontoblast processes. The present study demonstrated that nerve endings (varicosities) derived from the trigeminal ganglion were present in the dentinal tubules, and that WGA-HRP extracellularly extruded from the sensory nerves in the odontoblastic layer or predentin/dentin. These findings thus suggest that sensory nerves may have some (e.g., trophic) effect on either odontoblasts or the environment around the sensory nerves in the dentin/pulp.     

A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.     

CMAP: a novel cystatin-like gene involved in liver metastasis

The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)-DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.     

Histogenesis of the chequered pattern of ivory of the African elephant (Loxodonta africana)

This study aimed to propose a hypothesis on the events which lead to the development of the characteristic chequered pattern of elephant ivory. Twenty fragments of ivory and six elephant tusks were obtained through the National Parks Board of South Africa. Polished surfaces were prepared in sagittal and longitudinal planes and the characteristics of the distinctive chequered pattern described. Light- and electron-microscopical techniques and image analyses were employed to determine the morphological basis of the pattern and to describe the spatial distribution, density and morphology of the dentinal tubules. These investigations showed that the distinctive pattern was the result of the sinusoidal, centripetal course followed by dentinal tubules. The apical, slanted part of the sinusoidal curve is the result of the centripetally moving odontoblast, which, during formation of ivory, progresses towards the centre of the tusk on a decreasing circumference. It is suggested that this leads to cell crowding, increased pressure between odontoblasts and subsequent apical movement of their cell bodies, cell degeneration and fusion. Odontoblastic degeneration and fusion probably relieve the pressure between the crowded odontoblasts by reducing their numbers and the remaining odontoblasts now orientate their centripetal course towards the tip of the tusk, thereby forming the anterior-directed part of the sinusoidal path of the tubule. As odontoblasts progress centripetally the diameter of the pulpal cavity decreases further and the processes of apical movement, fusion and degeneration of odontoblasts are repeated. This occurs until the pulpal cavity is obliterated.     

Elemental distributions in predentine associated with dentine mineralization in rat incisor

Electron probe microanalysis was applied to study quantitatively and semi quantitatively the elemental concentrations and distributions that occur in predentine during the dentine mineralization of rat incisor. Apex regions of the continuously growing incisors were rapidly dissected and cryofixed in liquid nitrogen-cooled propane. Ultrathin cryosections were prepared from the dentine tissue. On the average in the extracellular predentine element concentrations of calcium and phosphorus were about 0.5% (w/w) and 0.5-1% (w/w), respectively; so the calcium content in the extracellular predentine is higher while the phosphorus content is much lower than in the odontoblast area. Due to the high content of glycosaminoglycans in the extracellular matrix the concentration of sulfur in the predentine was more than 1% (w/w); the potassium content was found in the range of 0.6-0.8% (w/w) which is quite high for an extracellular area and the concentrations of sodium and chlorine were higher than 2% (w/w). Elemental mapping analysis was carried out to demonstrate the distribution of some important elements at the predentine/dentine border during mineralization.     

New horizons for therapy based on the boron neutron capture reaction

Experimental studies were undertaken in immature rat molar teeth to investigate the relationship between root development and the prognosis after partial extrusion. The experimental partial extrusion was induced at 1/2 root formation, 2/3 root formation and 3/4 root formation, using an extrusion device designed by the author. Histopathological studies and SEM observation of the pulpal tissue in rat maxillary first molars with partially formed roots after partial extrusion were conducted and evaluated. The degree of extrusion was changed according to the development of the root to produce a similar luxated state. The rats were sacrificed immediately after experimental extrusion, immediately after operation, and after 3 days, 7 days, 14 days, 30 days, and 60 days to compare the histopathological and SEM findings between experimental groups and control groups. Immediately after the extrusive injury, ablations of the odontoblast layer from dentin were seen with retention of a fluid component in the ablated spaces in the pulp. On 1 and 3 days after surgery, cellular components such as erythrocytes and leukocytes due to bleeding were seen. Ablations of the odontoblast layer were seen in the coronal pulp most frequently in the pulp horn, cervical area and floor of the pulp chamber. In general, pulp reaction after extrusion in groups with more complete root formation was more severe than in groups with less complete root formation. It is suggested that the more complete the root formation, the greater the effect on the pulp as result of partial extrusion, while with less complete root formation, the greater the malformation of the root apices.     

In situ localization and chromosomal mapping of the AG1 (Dmp1) gene

Dentinogenesis is being used as a model for understanding the biomineralization process. The odontoblasts synthesize a structural matrix comprised of Type I collagen fibrils which define the basic architecture of the tissue. The odontoblasts also synthesize and deliver a number of dentin-specific acidic macromolecules into the extracellular compartment. These acidic macromolecules may be involved in regulating the ordered deposition of hydroxyapatite crystals within the matrix. AG1 is the first tooth-specific acidic macromolecule to have been cloned and sequenced. To identify which cells of the rat incisor pulp/odontoblast complex were responsible for synthesis of AG1, in situ hybridization was used. Digoxigenin labeled sense and anti-sense AG1 riboprobes were prepared. The AG1 mRNA was found to be expressed in the mature secretory odontoblasts. Neither pulp cells nor pre-odontoblasts showed any staining with the anti-sense probes. Chromosomal localization studies placed the AG1 gene on mouse chromosome 5q21, in tight linkage with Fgf5. AG1 has been renamed Dmp1 (dentin matrix protein 1) in accordance with present chromosomal nomenclature. Mouse 5q21 corresponds to the 4q21 locus in humans. This is the locus for the human tooth mineralization disorder dentinogenesis imperfecta Type II (DI-II). These data suggest that the Dmp1 gene is involved in mineralization and is a candidate gene for DI-II.     

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.     

A light microscopic study of odontoblastic and non-odontoblastic cells involved in tertiary dentinogenesis in well-defined cavitated carious lesions

This study examines cellular and microradiographic findings in thin, undemineralized sections of 46 cavitated lesions, that were clinically well-defined with respect to lesion activity and estimated lesion age at extraction time. The progressive stages of surface breakdown ranged from enamel cavitation to larger dentine exposures classified as closed and open lesion environments. Measurements of the following parameters were performed using computerized image processing software: (a) the cytoplasm:nucleus ratio of primary odontoblast cells; (b) the cell:dentinal tubule ratio; (c) the adjacent predentine area (mum2), and (d) the cytoplasm: nucleus ratio of non-odontoblastic cells, and secondary odontoblast-like cells, where estimation of these cell types were based on structural criteria. In active enamel cavitated lesions, reduced odontoblast-predentine regions and indistinct subodontoblastic regions were noted. During initial dentine exposures, non-odontoblastic cells along the pulp-dentinal interface were observed as well. The first indication of tertiary dentine was seen in old lesions with exposed dentine. The tertiary dentine appeared more atubular in the closed/active lesions than in the open/slow-progressing lesions. The involved odontoblastic cells in tubular tertiary dentine in small open/slow-progressing lesions were comparable to the primary odontoblast cells, however, new dentinal tubules were also noted presenting a mixture between reactionary and reparative dentinogenesis. In close/active lesions non-primary odontoblastic cells were aligning the atubular tertiary dentine, whereas well-defined signs of secondary odontoblast-like cells were first seen in larger open lesions, producing tubular tertiary dentine. In conclusion, a strong relationship between external lesion environments and corresponding different formations of tertiary dentine was noted in advanced cavitated lesions. It is additionally suggested that the stimulation of tubular tertiary dentine could be a closely related reaction when an active lesion complex changes into a slower progressing lesion environment.     

Cloned 3T6 cell line from CD-1 mouse fetal molar dental papillae

Only primary pulpal cell cultures and one virally transformed mouse cell culture have been formally reported in the literature to synthesize proteins such as phosphophoryn which are unique to dentin matrix. In the present study, a mixed culture was derived from dental papilla cells of 18-19 fetal day CD-1 mouse mandibular first molars, maintained on a 3T6 plating regimen, and subsequently cloned after 28 passages. This cloned cell line (MDPC-23) exhibited several unique features, some of which were characteristic of odontoblasts in vivo. The features of this cell line included (1) epithelioid morphology of all cells with multiple cell membrane processes, (2) high alkaline phosphatase activity in all cells, (3) formation of multilayered nodules and multilayered cultures when maintained in ascorbic acid and beta-glycerophosphate, and (4) expression of two markers for odontoblast differentiation, i.e. dentin phosphoprotein and dentin sialoprotein.     

Ritual head computed tomography may unnecessarily delay lifesaving trauma care

This study compared the tensile bond strengths of soft lining materials polymerized by using conventional water bath methods and microwave energy. The soft lining materials used in this investigation were heat temperature vulcanizing (H.T.V.) silicon material Molloplast-B, and room temperature vulcanizing (R.T.V) acrylic material Getz Soft Oryl. The H.T.V. specimens were prepared with retention, additional retention and without retention, and the R.T.V. specimens were prepared by using bonding agent and bonding agent plus retention. All were cured by both conventional water bath and microwave energy. Results showed that the mean bond strength of H.T.V. specimens ranged from 9.6 to 13.12 kg/cm2, while the mean bond strength of R.T.V. specimens ranged from 0.36 to 1.75 kg/cm2. In both conventional and microwave groups, the specimens prepared with retention, additional retention and without retention or using bonding agent and bonding agent plus retention did not show any significant difference when they were compared separately. But the difference between conventional and microwave groups was found significant in both H.T.V. and R.T.V. specimens. It showed that conventional water bath technique is better than microwave technique.     

Effects of xylitol and carbohydrate diets on dental caries, dentine formation and mineralization in young rats

Female Wistar rats were weaned at the age of 3 weeks and fed for 7 weeks either a high-sucrose diet, a non-cariogenic raw potato-starch diet, a high-sucrose diet with 5% xylitol supplement, a raw potato-starch diet with 5% xylitol supplement or a non-cariogenic, commercial, powdered rat food (Ewos R3) for reference. A low xylitol concentration reduced the progression and severity of carious lesions but did not affect dentine apposition or the width of predentine in rats fed high-carbohydrate diets. Widening of the predentine zone in rats fed a high-sucrose diet might reflect disturbed mineralization, which could not be explained by serum ionized calcium or phosphate ion levels and which could not be corrected by low xylitol concentrations. It is concluded that the reduced area of dentinal carious lesions after low xylitol supplementation is not dependent on dentine formation or mineralization, but rather on direct effects in the mouth.     

Pulp response in primary teeth with deep residual caries treated with silver fluoride and glass ionomer cement ('atraumatic' technique)

Histological assessment of the dental pulps of 55 carious primary teeth was carried out 3 to 58 months after treatment by the 'atraumatic' technique involving application of 40 per cent silver fluoride to residual caries followed by restoration with glass ionomer cement. Fifty of the 55 teeth examined showed a favourable pulpal response, inducing presence of abundant reparative dentine and a wide odontoblast layer. Histological comparisons were made between these teeth and others not treated with silver fluoride but restored with glass ionomer cement, amalgam or zinc oxide and eugenol. Possible mechanisms of the action of silver fluoride in arresting residual caries are discussed. The question of whether or not treatment of carious dentine with silver fluoride represents a biologically acceptable clinical procedure cannot be answered on the basis of pulpal histology alone. The very high concentration of fluoride in commercial preparations of silver fluoride raises several questions concerning its clinical safety.     

Effects of CO2 laser irradiation in vivo on rat alveolar bone and incisor enamel, dentin, and pulp

Previous studies have shown that a surgical 'window' can be drilled in alveolar bone for experimental manipulation of the underlying enamel organ and enamel. To determine whether similar and/or improved access could be obtained by use of the surgical capabilities of laser optics, and to note the effects of laser irradiation in vivo on the extracellular matrices and cells of bone, enamel, and dentin, tissue responses to laser-created lesions were examined histologically. Briefly, samples were prepared in which the alveolar bone along the inferior mandibular border of Wistar rats was exposed, and a continuous-wave CO2 laser equipped with a custom-made micromanipulator was used to penetrate the bone and to create lesions within the lower incisor. Animals were perfusion-fixed at either 10 min or 10 days post-treatment, and affected tissues were processed for light and transmission electron microscopy. At 10 min, all lesions consisted of a void of ablated tissue containing some organic debris. Tissues immediately surrounding the lesion were generally intact, but showed some damage, presumably resulting from elevated temperature effects. At 10 days, lesions in the bone, dentin, odontoblast layer, or pulp showed morphological evidence of tissue repair represented by the presence of cell infiltrates, new bone, or reparative dentin. In lesions that were created during the secretory stage of amelogenesis that had moved into the maturation stage, there was evidence of delayed or incomplete maturation of enamel (i.e., retention of organic matrix normally lost during maturation) related to the enamel organ affected by the laser treatment. In the bone lesion at 10 days, new bone formation was observed, while bone fragments originally created at the time of lasing were surrounded by mononuclear and large multinucleated giant cells. It is thus concluded that the application of this laser system is an alternative method for exposing unerupted dental tissues for experimental manipulation, and that laser irradiation may also be useful for the study of mineralized tissue repair.     

Preoperative evaluation of the chemosensitivity of breast cancer by means of double phase 99mTc-MIBI scintimammography

The chemosensitivity of breast cancer is important for its management, but it is difficult to evaluate preoperatively. Tc-99m hexakis-2-methoxyisobutylisonitrile (MIBI) scintimammography has been reported to indicate the expression of P-glycoprotein, which is one factor concerned with multidrug resistance. We developed a chemosensitivity assay by using surgical specimens to investigate whether 99mTc-MIBI scintimammography findings before the operation are related to chemosensitivity according to our assay. Fifteen patients with primary breast cancer were enrolled into the study. Early and delayed images were obtained at 10 and 120 minutes after intravenous injection of 99mTc-MIBI, respectively. Regions of interest were placed on the tumors and the contralateral healthy breasts in each patient to estimate 99mTc-MIBI uptake in the tumor, and retention indices were then calculated to assess the washout of 99mTc-MIBI. Chemosensitivity assay was performed by incubating surgical specimens with anticancer agents such as doxorubicin, epirubicin, pinorubicin, mitomycin C, cisplatin and 5-fluorouracil. 99mTc-MIBI washout on scintimammography was successfully related to inhibition ratios on chemosensitivity tests when compared with 99mTc-MIBI uptake by the tumor. In particular, high correlation coefficients were obtained between the retention index of 99mTc-MIBI and the inhibition ratios of doxorubicin (r = 0.75), epirubicin (r = 0.60) and pinorubicin (r = 0.62), but poor correlation was found for mitomycin C (r = 0.44) and cisplatin (r = 0.31). Our results indicate that the retention index of 99mTc-MIBI is closely correlated to chemosensitivity to anthracyclines, suggesting that double-phase scintimammography allows preoperative prediction of chemosensitivity of breast cancer.     

Distribution and organization of odontoblast processes in human dentin

The distribution and organization of odontoblast processes in young human dentin was examined with a scanning electron microscope. By applying the HCl-collagenase method, the extracellular matrix of dentin was almost completely removed, thereby exposing the odontoblast processes and their branches to direct observation. The odontoblast processes are located close to the dentinoenamel junction. In the middle and outer zones of dentin the processes bear numerous branches. Some of these appeared to bridge the space between the processes and connect them.