Subodontoblastic Layer Research Articles

In situ localization of tenascin mRNA in developing mouse teeth

Tenascin is a large extracellular-matrix glycoprotein found in developing connective tissue. A cDNA probe to mouse tenascin, mTN2, was used to determine the cellular origins of this molecule in the murine tooth germ by in situ hybridization. At embryonic day 19, a hybridization signal significantly greater than background was detected with mTN2 in the subodontoblastic layer of the dental mesenchyme and in the inner enamel epithelium of the enamel organ. At postnatal day 1, a signal was detected over pre-odontoblasts and the strata intermedium and externum. No tenascin mRNA was detected in odontoblasts or the stellate reticulum at either age, and hybridization in ameloblasts was not significantly greater than background at postnatal day 1. Thus, much of the tenascin found throughout developing teeth appears to be synthesized by pre-odontoblasts and the inner enamel epithelium, the two populations of cells destined to generate mineralized matrix.

A Chondroitin Sulfate Epitope in Mammalian Dental Pulp and its Developmental Expression in Mouse Dental Papilla

The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.

Adriamycin alters the alkaline phosphatase activity in hamster molars during development in vitro.

The effect of a 2 hour exposure to adriamycin (1 mg/litre) on alkaline phosphatase (ALPase) activity of the golden hamster 4-5 day old second maxillary molars (M2) was investigated in vitro. The molars were grown in BGJb medium containing 15% fetal bovine serum, glutamine (200 micrograms/ml), vitamin C (250 micrograms/ml), penicillin G (50 micrograms/ml), and streptomycin sulphate (30 micrograms/ml). The gas phase contained 50% O2 + 5% CO2 + 45% N2. The molars were supported on cellulosic membrane filters and grown for 3, 5, and 7 days at the medium-gas interface in a closed humidified chamber. Biochemical analysis indicated a steady increase in ALPase activity throughout this study in the control samples. However, after adriamycin treatment no increase in ALPase activity could be observed. The histochemical data showed that the increased activity in the control was confined to the peripheral pulp, sub-odontoblastic layer, stratum intermedium, ameloblasts and odontoblasts. Although these layers showed a decreased activity after adriamycin treatment, the ameloblasts showed an increase in activity over the control. The data has shown that adriamycin caused a reduction in total ALPase activity in developing molars in vitro; osteodentin production by pulp cells; and appeared to produce an acceleration in the differentiation of ameloblasts.

Investigation of the role of Von Korff fibers during murine dentinogenesis: Bhavna Shroff Farmington, Connecticut: University of Connecticut, 1989

The existence and significance of Von Korff fibers during early dentinogenesis are still very controversial. The purpose of the present study was to re-examine the questions of the existence, nature and significance of Von Korff's fibers using light microscopy and immunohistochemistry. Specimens were obtained from 3 days-old CD-I mice and mandibles were carefully dissected under constant irrigation and immediately fixed in 10% neutral buffered formalin for light microscopy. Sections were treated or not with collagenase prior to silver staining. For immunohistochemistry, specimens were fixed in 95% ethanol and embedded in paraffin. Sections were reacted with goat anti-human-bovine type I or type III collagen and a rhodamine (RITC) labelled rabbit anti-goat IgG was then reacted as a secondary antibody. Slides were then examined under a Zeiss II photomicroscope equipped with epifluorescence. Our results have confirmed the presence of argyrophilic material concentrated at the periphery of the dental papilla and stretching from the subodontoblastic layer to the future dentino enamel junction. The distribution of type III collagen was very similar to the distribution of the silver staining at the cervical loop area. Type I collagen distribution was different and concentrated in areas where odontoblasts were fully differentiated. Our study showed that Von Korff fibers are not artefactual. We have established the presence of an apical compartment containing type I collagen fibers and a basal compartment containing type III collagen to explain the image of continuous silver staining crossing the entire thickness of the odontoblast layer.

Localization of alkaline phosphatase in developing bovine dental pulp

The distribution of alkaline phosphatase in dentinogenically active bovine dental pulp tissues was investigated histochemically and biochemically. Histochemical observation showed a high enzyme activity in the subodontoblastic layer, especially in coronal pulp and also in the core of radicular pulp. Biochemical results were well consistent with histochemical findings. Alkaline phosphatase activity in the coronal pulp was twice that in radicular pulp. Most of the enzyme activity in coronal pulp was in the subodontoblastic layer (91%), whereas the activity in the radicular pulp was evenly distributed. Since undifferentiated mesenchymal cells were observed in the areas showing relatively high ALPase activity, it seemed that there were some relations between the inducion of ALPase and the undifferentiated mesenchymal cells.

Lysyl-oxidase activity in the odontoblast-predentine layer isolated from bovine incisor teeth

This layer had lysyl-oxidase (EC 1.4.3.13) activity, 4–11 times higher than either the sub-odontoblast layer or central pulp tissue, and similar to that in chick aorta, one of the tissues richest in such activity. This finding relates to the potential for rapid cross-link formation following synthesis of the predentine collagenous matrix.

Cyclophosphamide-induced changes in rodent odontogenesis. A light- and electron-microscopic study.

Cyclophosphamide-induced changes in rodent odontogenesis were investigated by light and electron microscopy in four-day-old Sprague Dawley rats given one injection of 40 mg/kg of body weight of cyclophosphamide and killed at intervals of one hour, one day, one week and two weeks. Incisor and molar teeth were dissected from the animals, fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate with 3.4% sucrose, and subsequently some were incubated for alkaline phosphatase reaction, and embedded in Spurr's medium for sectioning at light- and electron-microscopic levels. From three days a cell-sparse zone was created in the pulp in the growing end of the tooth and progressive cellular changes were observed which became more severe in the one-week and two-week specimens. Subodontoblast and adjacent pulpal cells were the most affected showing nuclear changes, damage to, or loss of, organelles, and inclusion bodies. Odontogenic epithelium was less affected and odontoblasts appeared to be unaffected by the drug. A new irregular matrix was laid down in the defect area and seemed to be the product of depolarized odontoblasts. This new matrix showed alkaline phosphatase activity, as did the cells embedded in it, and later it became mineralized. It is speculated that the polarity of odontoblasts might be maintained by an intact subodontoblastic layer; when this is lost the odontoblasts become depolarized and capable of secreting matrix from both ends.

Histological study of pulps of human primary teeth

Twenty-seven primary human canines and incisors showing no clinical signs of caries, root resorption or restorative treatment were extracted for orthodontic purposes from patients ranging in age from 7 to 13 1 2 yr , and processed for histologic examination. Odontoblasts were pseudostratified in depths of 1–7 cells in a pulpward direction, with the thickest layers in the coronal pulp. The zone of Weil and the cell-rich zone were present in the coronal third of normal pulp, but absent in teeth with occlusal attrition. Vascular trunks were oriented coronally and exhibited increased branching as they approached the subodontoblastic layer. Collagenous fibers appeared coarse, densely packed, and coronally oriented in the apical third of the pulp, whereas the middle and coronal fibres were fine, loosely packed and randomly oriented. A cap-like dense zone of collagenous and reticular fibres was noted in pulp horns and the periphery of the coronal pulp. Von Korff's fibres were well developed in the coronal pulp but only poorly developed in apical and middle regions of the pulp. Focal mineralizations, closed apices, and a coronal-to-apical change in the staining characteristics of the predentine-odontoblastic border were also noted. There was no structural difference between primary human pulp and young permanent human pulp, other than the presence of a cap-like zone of reticular and collagenous fibres in primary coronal pulp.

Cholinesterases and choline acetylase in isolated human and rat odontoblasts

Activity and localization of cholinesterases and activity of choline acetyltransferase was determined in isolated rat and human odontoblasts. The hydrolysis of choline esters was measured by the microdiver and radiometric assays and the activity of choline acetyltransferase by a radiometric assay. The cholinesterase activity in individual odontoblasts of man and rat was almost the same and amounted in man to 24 pmol hydrolysed substrate/odontoblast per h if 3.3 mM acetylcholine was used, and in the rat it amounted to 27 pmol hydrolysed substrate/odontoblast per h at the same concentration of acetylcholine. In odontoblast homogenates, the activity of choline acetyltransferase amounted to 6 nmol ACh/g of fresh tissue per min. Histochemical and electron-microscopic studies showed the cholinesterase localized in the subodontoblast layer and in the nucleus of the odontoblast. The presence of cholinesterase and choline acetyltransferase in odontoblasts suggests that odontoblasts possess some features of a cholinergic system.

Histochemical characterization of alkaline phosphatase in developing rat teeth and bone

Alkaline phosphatase (EC 3.1.3.1.) in developing teeth and in bone has been studied. Prior to hard tissue function a rather high enzyme activity was noted in differentiating odontoblasts, stratum intermedium, and outer enamel epithelium. A lower activity was observed in the cells of the dental papilla and stellate reticulum. After the onset of hard tissue formation the alkaline phosphatase activity was generally increased. Enzyme activity was also found in the proximal part of tall, secretory ameloblasts. In the short postsecretory ameloblasts a high enzyme activity was noted. At the onset of dentin mineralization there was an increase in enzyme activity in the cells of the subodontoblastic layer. In bone the highest alkaline phosphatase activity was found in osteoblasts. A difference was noted between the alkaline phosphatase of hard and soft tissues by means of the addition of inhibitors to the incubation media. Within the hard tissues it was possible to distinguish between two alkaline phosphatases after pretreatment with heat (56 degrees C) or the addition of specific inhibitors (sodium metavandate, ortho-and pyrophosphate). An isoenzyme which was sensitive to these procedures was demonstrated in odontoblasts and in the pulpal connective tissue. Another alkaline phosphatase isoenzyme, which was resistant to pretreatment with heat or the addition of vanadate or phosphate, was demonstrated in the subodontoblastic cell layer, stratum intermedium and the outer cells of the reduced enamel epithelium.

Alkaline phosphatase in developing teeth and bone of man and macaque monkey.

The activity of nonspecific alkaline phosphatase (E.C. 3.1.3.1) in developing teeth and bone of human fetuses and young macaque monkeys has been studied by means of histochemistry. The incubations for alkaline phosphatase were performed at pH 8.2 using naphthol-AS-MX-phosphate as substrate and Fast Blue RR salt or Fast Red Violet LB salt as couplers. By means of pretreatment with heat (56 degrees C), or addition of sodium metavanadate, ortho- or pyrophosphate, two alkaline phosphatases were demonstrated in the developing teeth. Prior to hard tissue formation all alkaline phosphatase activity was inhibited by the addition of vanadate, phosphate, or by pretreatment with heat. Pretreatment with heat or addition of vanadate or phosphate also inhibited alkaline phosphatase activity in the odontoblasts and in the pulpal connective tissue, whereas the activity in the subodontoblastic cell layer, stratum intermedium, outer enamel epithelium, and the the outer cells of the reduced enamel epithelium were much less affected. A week resistant activity was also noted in odontoblasts and pulpal connective tissue.

Enzyme activity in the pulp following preparation of cavities and insertion of medicaments in cavities in monkey teeth

The effect of cavity preparation, calcium hydroxide and a corticosteroid on pulpal enzymes (Alkaline phosphatase, acid phosphatase, beta-glucuronidase, cytochrome oxidase and succinate, lactate and glucose-6-phosphate dehydrogenase) in monkey teeth has been studied by histochemical means. Cavity preparation with an air turbine and sufficient spray apparently did not affect the enzyme activity of the pulp, nor did application of a corticosteroid to the cavity floor. Twenty-four hours after calcium hydroxide application an increase in enzyme activity was found in the ondontoblastic and subodontoblastic cell layers subjacent to the calcium hydroxide-covered dentin. This activity seemed to demonstrate an onset of dentin formation, and 15 days after the application a slight amount of secondary dentin was found subjacent to the cavities in these teeth.

Adenosine triphosphate hydrolysis in rat dental tissues

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.

Alkaline phosphatase, 5?-nucleotidase and ATPase activity in the molar region of the mouse

The distribution of nonspecific alkaline phosphatase (EC 3.1.3.1), ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5) was studied by histochemical calcium and lead methods in the molar region of newborn mice. Glycerophosphate (GP), adenosine triphosphate (ATP) and adenosine monophosphate (AMP) were tested as enzyme substrates. In order to distinguish between hydrolysis catalyzed by nonspecific alkaline phosphatase and hydrolysis mediated by more substrate specific phosphatases, inhibitors of different phosphatase activities were added to the incubation media. Alkaline phosphatase (EC 3.1.3.1), assayed as GPase in osteoblasts, cells of the stratum intermedium, odontoblasts, cells of the subodontoblastic layer and blood vessel walls was completely inhibited by R 8231, BAL and EDTA and almost completely inhibited by L-cysteine and levamisole. The AMP-hydrolyzing capacity in the cells mentioned above was identically affected and was considered to be due to nonspecific alkaline phosphatase. In the oral epithelium and the external dental epithelium, on the other hand, the AMPase activity recorded with the lead method (pH 7.2) was not affected by these inhibitors while Zn2+ ions caused inhibition. In view of these findings the AMP-hydrolysis in oral epithelium and external dental epithelium was considered to be due to 5′-nucleotidase (EC 3.1.3.5). The ATPase activity in ameloblasts, tongue muscle fibres and blood vessel walls was unaffected by R 8231 and levamisole and enhanced by BAL and L-cysteine. Treatment with EDTA increased the ATPase activity recorded in muscle fibres and blood vessel walls. The major part of the ATP-hydrolyzing capacity in osteoblasts, cells of the stratum intermedium and odontoblasts was inhibited by R 8231 BAL and EDTA. The hydrolysis of ATP recorded after inhibition by R 8231, BAL or EDTA is likely to be due to activity of substrate-specific ATPase(s) (EC 3.6.1.3).

A histochemical study of arylaminopeptidases and alkaline phosphatases in sound and carious human teeth.

Fresh, undecalcified sections (of 6—20 μm) from intact or carious human teeth as well as teeth with different types of pulpal inflammation were cut in a heavy duty microtome at —20° C. Scotch tape was used to stabilize the teeth during sectioning. The localization of arylaminopeptidases (EC 3.4.1.) and alkaline phosphatase (EC 3.1.3.1.) was demonstrated histochemically using an azo dye principle. The teeth were also examined by microradiography. No arylaminopeptidase activity was normally observed in intact teeth by the histochemical method. In carious teeth, however, this enzyme activity could be localized in the dentinal tubules of the carious lesion, whereas sound dentin, predentin and pulp did not reveal any activity. The inflamed part of the pulp was observed to reveal marked arylaminopeptidase activity. Alkaline phosphatase was observed to be localized mainly in the predentin layer and in the subodontoblastic layer of the pulp and additionally in the cementum and in the remaining parts of the period...

A light and electron microscopic study of alkaline phosphatase activity in the early stage of dentinogenesis in the young rat

Alkaline phosphatase activity in the early stage of dentinogenesis of 1-day-old rat teeth was examined histochemically by means of both light and electron microscopy. The enzyme activity appearing to be associated with von Korff fibres under light microscope was revealed by the electron microscope to be activity associated not only with fibrillar elements but also with extracellular amorphous ground substance. The activity in odontoblasts and in sub-odontoblastic pulp cells occurred mostly in association with cell membranes. A few granules at the peripheries of these cells may indicate slight activity. Enzyme diffusely distributed in the sub-odontoblastic layer under light microscopy proved by electron microscopy to be associated with intercellular amorphous ground substance as well as cell membranes. The possible role of alkaline phosphatase in the early stage of dentinogenesis is discussed.

Age changes of the vascular pattern of the human dental pulp

A study is presented for the purpose of showing the change in vascular architecture of the human dental pulp with age. It is shown that as age increases, the number of vascular structures decreases, primarily at the expense of vessel reduction in the sub-odontoblastic layer of the pulp. It is demonstrated that the human pulp has a natural arterio-venous shunt system.

The distribution of alkaline phosphatase in the human tooth germ

The distribution is reported of alkaline phosphatase in the human tooth germ from the time of initiation up to and including the stage where the first increments of enamel matrix have formed. The early occurrence of enzyme activity in the stellate reticulum is considered to be associated with the determination of the crown pattern of the tooth. The gradient of enzyme activity in the components of the enamel organ is thought to reflect metabolic activity. Particular attention is drawn to the presence of cell layers rich in alkaline phosphatase, the sub-odontoblast layer and the stratum intermedium. The significance of this finding in relation to the production of calcified tissues is discussed. The absence of alkaline phosphatase activity in odontoblasts and ameloblasts is reported and discussed in relation to current views on hard tissue genesis.

Aspects histochimiques du developpement des bourgeons dentaires

A histochemical study of the tooth germs of mice has demonstrated the precise sites of succinic dehydrogenase and of an enzyme dephosphorylating adenosine triphosphate, adenosine diphosphate and adenosine monophosphate. The two enzymes are present in the odontoblast and subodontoblastic layers of the pulp and in the inner cellular layer of the enamel organ; their cytoplasmic localization is quite similar. The intensity of the reactions brought about by the enzymes continues to increase during the development of the tooth germs and reaches a maximum when mineralization takes place. Their participation in the processes of mineralization can be regarded as a system for draining phosphate ions to the dentine and to the enamel.