Calcified Dentin Research Articles

Transcriptional Regulation of Dentin Matrix Protein 1 (DMP1) by AP-1 (c-fos/c-jun) Factors

Dentin matrix protein 1 (DMP1) is an extracellular matrix phosphoprotein whose expression is precisely controlled temporally and spatially during the differentiation of the odontoblasts. Odontoblasts are postmitotic cells that differentiate to mature polarized cells and are responsible for the synthesis of a calcified dentin matrix. Although the DMP1 promoter has been isolated from the mouse and the rat, little is actually known about their control by specific transcription factors. Analysis of the rat DMP1 promoter has identified several regulatory transcription factors. These factors include AP-1 family members, SP1 and ETS. Therefore the transcription of DMP1 may be controlled positively or negatively by a variety of stimuli resulting in a developmentally regulated gene expression. This study demonstrates the role of c-fos and c-jun on the transcriptional regulation of DMP1 gene.

Pedicellate Teeth and the Problems of Amphibian Phylogeny

Living amphibians are the only group of Recent tet-rapods possessing so-called pedicellate teeth. There-fore, amphibian dentition attracts considerable atten-tion of morphologists and histologists. The studies onthe structure and development of individual pedicellateteeth of anurans and caudates have shown that the pedi-cellate teeth have a complex structure and consist oftwo calcified dentin elements, the pedicel and thecrown, connected to each other by a nonmineralizeddividing zone [1–4]. The study on the ontogeny of thedental system has revealed that each out of three extantamphibian orders are characterized by complexchanges of dentition until the stable definitive state isreached. Thus, in caudates, three morphological typesof teeth successively replace each other; anurans pos-sess two types distinguished mainly by the presence orabsence of the dividing zone [5–7]. In the order Gym-nophiona, the transformation consists mainly in thechanges of the crown shape of pedicellate teeth [6].The purpose of the present study was to analyze thedevelopment of teeth of different morphological typesin various groups and the formation of the dividingzone, since these aspects of tooth development remainlargely unknown.In addition to the interest in purely morphologicaland comparative evolutionary problems, this task isimportant for gaining an insight into such extensive andcomplex questions as the evolution of the entireamphibian class and the validity of the taxon Lissam-phibia.We examined age series of representatives of thethree extant amphibian orders (seven species of cau-dates, nine anuran and one apodan species). The prepa-rations were produced by staining the material fixed in10% formalin with alcohol solution of alizarin red S, sothat calcified tissue became red.The study on the development of the dental systemfrom the beginning of its formation (at the stages ofhatching in caudates and at the final stages of metamor-phosis or in postmetamorphosis in anurans) hasrevealed that, typically, members of both orders showsuccessive replacement of the nonpedicellate teethcharacteristic of early developmental stages by thepedicellate teeth of the late stages. These tooth genera-tions are distinguished not only by the morphologicalstructure (mainly by the presence or absence of thenonmineralized dividing zone between the crown andthe pedicel), but also by the calcification pattern.The calcification of a young developing nonpedicel-late tooth is initiated at the apical part and extends uni-directionally to the tooth base; subsequently, a toothfuses with the dentigerous bone and achieves the func-tional state. The calcification of pedicellate teeth alsobegins from the crown apex and proceeds proximally.However, this process abruptly stops approximately inthe middle of the height of the future mature tooth; sub-sequently, a second center of calcification is formed onthe external (labial) side of the nonmineralized pedicelproximal to the boundary of the calcified tissue. Inyoung teeth stained with alizarin, the second centerappears as a pink spot extending in parallel to the divid-ing zone formed in the tooth. Subsequently, the calcifi-cation extends from this center to the base and involvesfirst the labial part of the pedicel; then, the lateral part;and, finally, the lingual part. When the calcification ofthe pedicel is completed, the tooth becomes rigidlyattached to the bone and achieves the functional state;only the dividing zone remains nonmineralized.In some cases, in amphibians of either order, thedividing zone is also calcified to a greater or lesserextent. The generation of so-called subpedicellateteeth, distinctive of caudates and characteristic of thelate larval stages immediately before the metamorpho-sis, initially follows the same way of development astrue pedicellate teeth do, i.e., the calcification of thecrown and pedicel begins in two separate centers. How-ever, along with the tooth maturation, the dividing zonealso becomes completely calcified; thus, when thefunctional state is achieved, the zone becomes almostindistinguishable in stained preparations.Complete mineralization of the dividing zone anddevelopment of secondary nonpedicellate teeth werealso observed in anurans. For example, this is typical of Pyxicephalus adspersus, Conraua crassipes (Ranidae)[7], and old functional teeth of all adult anurans. On thecontrary, in some cases, the labial part of the pedicel is

Archaeological evidence for the extinction of a breeding population of elephant seals in Tasmania in prehistoric times

Southern elephant seals, Mirounga leonina L., do not inhabit the northwest coast of Tasmania today, but archaeological evidence indicates that they did so in prehistoric times, when they constituted an important food resource to the Aboriginal tribes of the region. Skeletal remains of at least 300 elephant seals were present in one midden alone. There is distinct sexual dimorphism in the canine teeth of elephant seals, and regular seasonal variations in the density of concentric layers of calcified dentine, as well as the pattern of these variations, provide insight into the age and reproductive history of individual animals. The sectioned canine teeth of 145 southern elephant seals (107 females, 38 males) from a Tasmanian midden were examined to provide information on the age and sex of the seals as well as aspects of their reproductive history. The age distributions differed between the sexes, and partly explain the different frequencies of males and females. All the males were young, immature individuals, none more than 6 years old, which is about the age at which a secondary growth spurt occurs in males and results in a marked sexual disparity in body size. By contrast, 47% of the females were of breeding age, 26% had given birth to pups, and several were up to 20 years of age. At least 26% of animals were estimated to be less than 3 months old, the approximate age at which they go to sea for the first time, confirming that they were born on the northwest Tasmanian coast. Animals were killed throughout the year, and there is evidence of change in reproductive pattern over time, consistent with a response to predation pressure. The evidence points to the conclusion that the population was exterminated by Aboriginal hunters, through selective exploitation of smaller animals, which included significant numbers of breeding females. Copyright © 1999 John Wiley & Sons, Ltd.

CGRP immunohistochemistry in wound healing and dentin bridge formation following rat molar pulpotomy.

The aim of the present study was to investigate the neuropeptide CGRP in order to determine the effect on dentin bridge formation during the healing process after pulpotomy. First maxillary molars in 56-day-old Wistar rats (n=60) were used. The rats were killed for a neurohistopathological examination at 1, 3, 7, 14, and 28 days postoperatively. Neuronal changes in the residual pulp were studied using CGRP immunohistochemistry. By 1-3 days postoperatively, the CGRP-IR nerve fibers with abnormal beaded or knob-like structures were found to be more swollen than in the control and the leakage of a CGRP-IR-positive substance from the involved end of the nerve fibers was seen. At 7 days postoperatively, a vast number of newly sprouted CGRP-IR nerve fibers appeared in the residual pulp and some of them terminated in the differentiating odontoblast layer and the initial matrix layer of the dentin bridge. By 14-28 days, the nerve density had become progressively lower in the residual pulp. Regenerated axons also terminated in the odontoblast layer and the fibrous matrix layer of the calcified dentin bridge. These findings suggest that such sensory neuropeptides as CGRP may, therefore, play a role in dentin bridge formation in the rat molar.

Tissue reaction of hydroxyapatite sol to rat molar pulp.

Hydroxyapatite sol was prepared by dispersing unheated hydroxyapatite microcrystals into distilled water. A suspension of hydroxyapatite heated at 800 degrees C (HAp-800 degrees C) in distilled water was used as a comparative material. These were characterized by X-ray diffractometry, SEM and electroconductivity measurements. 0.001 ml of each material was injected into the upper first molar pulp of rats. After 7-14 days post-operatively, the group with hydroxyapatite sol showed slight inflammation in the pulp cavities and a highly calcified tubular dentine bridge was observed. Osteodentine had developed and adhered to the surface of the hydroxyapatite microcrystals. Matrix vesicles were observed in the extracellular matrix between collagen fibrils and electron-dense granules. After 28 days, the inflammation completely disappeared and tubular dentine was observed below the osteodentine. Acid phosphates (ACPase) activity was detected along all biosynthesizing pathways in odontoblast-like cells. Localized dehydrogenate (LDH) activity indicated phagocytosis of hydroxyapatite microcrystals by odontoblasts. In the group with HAp-800 degrees C after 7-14 days post-operatively, the inflammation was moderate. After 28 days, relatively fewer calcified dentine tubules were observed. ACPase activity was detected only along the membranes bounding cytoplasmatic vacuoles. It was concluded that hydroxyapatite sol is a safe biomaterial for use as dental pulp and induces early formation of dentine-bridge more so than HAp-800 degrees C.

Regulation of osteoclast function.

Regulation of osteoclast function.

Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts.

To examine the effect of extracellular matrix on osteoclast polarization, we focused on the actin organization in osteoclasts, using murine osteoclast-like multinucleated cells (OCLs) formed in cocultures of osteoblastic cells and bone marrow cells. When OCLs were cultured on either a plastic plate, calcified dentine, or calcium phosphate thin films in the presence of fetal bovine serum (FBS), they similarly formed ringed structures of F-actin dots (actin rings). However, OCLs placed on demineralized dentine or type I collagen gel matrix (collagen gel) failed to form actin rings. In the absence of FBS, actin ring formation in OCLs was induced on plastic plates coated with vitronectin, fibronectin, or type I collagen, but not on those coated with laminin, poly-L-lysine, or bovine serum albumin. Actin ring formation appeared to depend on integrins, since the GRGDS, but not the GRGES, peptide inhibited it in a dose-dependent manner. Moreover, immunoelectron microscopic examination revealed that vacuolar proton ATPase (V-ATPase) was localized along the apical membrane in much higher densities than the basolateral membrane in OCLs placed on plastic coverslips. In OCLs placed on collagen gel, however, V-ATPase was found to be distributed throughout the cytoplasm without polarity. These results suggest that actin ring formation in osteoclasts was dependent on matrix substrates, matrix proteins and integrins, and was closely related to osteoclast function.

Immunohistochemical localization of connexin 43 in the developing tooth germ of rat.

Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360-376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum intermedium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and predontoblasts was accumulated at cell surfaces facing the predentine.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures

The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Furthermore, a correlation between IL-1 alpha and prostaglandin E2 in the osteoclast recruitment induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide is discussed.

Dentine microstructure in hypoplastic deciduous teeth by collagenase etching

A new method is described for studying the microstructure of dentine by treating a polished surface with collagenase. The removal of collagen in this way allows high-resolution observation of the mineral component of dentine, and in this paper scanning electron micrographs are presented of dentine surfaces from , ‘normal’ teeth and caries-free hypoplastic teeth. In both cases, dentine is shown to consist of small rounded units ∼ 10–20 mn across which coalesce to form spheroids ∼ 100 nm in diameter which themselves aggregate to form a continuous mineral phase. The main difference between normal and hypoplastic teeth is that the inter-tubular dentine of the latter displays many more spaces, produced by the removal of greater numbers of collagen fibres from between the spheroidal particles. Thus the presence of less mineral would suggest that the carious process, once generated in such a tooth, may advance more rapidly than in normal teeth. The appearance of spheroids composed of smaller building units provides further evidence in support of the theory that calcification of dentine occurs at least in part by the apposition of mineral on the surface of calcospherites.

Bundle formation of principal fibers in rat molars.

The bundling of principal fibers was investigated in tangential sections through the tooth-related portion in developing rat molars by light and electron microscopy. When root dentin calcification began, cross sections of principal fibers emerged as fibril aggregates in the narrow intercellular spaces in a densely packed population of periodontal ligament cells. Subsequently, these cells changed shape and location to widen the intercellular spaces. The fibril aggregates became thicker in these spaces. With root development, the collagen fibrils formed loosely aggregated bundles and the periodontal ligament cells extended cell processes between the bundles. The cell processes usually contained microfilaments suggestive of actin filaments, and as the cell processes extended and came in close apposition, they formed delimited compartments. These compartments appeared to be a sheath-like structure, and the loose fibril bundles developed into tight fibril bundles in the compartments. Finally the principal fibers consisted of many tight fibril bundles, which were partially or entirely surrounded by cell processes and cell bodies. The findings suggest that the sheath-like, cellular compartments cause the tight bundling of the principal fibers.

EFFECT OF EHDP (1-HYDROXYETHANE-1, 1-DIPHOSPHONATE) ON RABBIT'S DENTINE CALCIFICATION FIGURES.

The effects of EHDP on dentine calcification figures in rabbits were examined by the lead acetate time marking method. The following results were obtained.1) It was confirmed by hematoxylin staining of the decalcified sections, X-ray analysis and microradiograms of the undecalcified sections and image analysis that EHDP causes hypocalcification in dentine calcification figures. The minimum dose of subcutaneous injection of 3 5 mg/kg (0.751.25 mgP/kg) produced the hypocalcifications and the degree of hypocalcification was strengthened in proportion to the dose.2) Subcutaneous injection of EHDP 60 mg/kg (14.9 mgP/kg) each day for seven consecutive days caused EHDP rickety rats. Hypocalcification dentine is formed within the administration period, and not only the growth rate of teeth but also the development of bone are decreased.3) One-hour injection into the arteria maxillaris or the arteria thyroidea caused hypocalcification dentine on the arterial infusion side at only 5 mg/kg, and strong formation on the same side and opposite side at 10 mg/kg and 30mg/kg. However, there were no specific changes in the thyroid tissues.4) A 30 mg/kg subcutaneous injection did not affect serum Ca, Ca2+, P or PTH concentrations. It is conjectured that the concentration of EHDP in the blood is related to the dentine calcification figures.5) From these results, the disturbance in dentine calcification that results from infusion at above doses can be conjectured to be related to the local action produced by the dentine formative function of EHDP.

EFFECTS OF ZINC ON RABBIT DENTINE CALCIFICATION AND ZINC CONCENTRATIONS IN SOME ORGANS.

Zinc is one of the essential elements in the living body, and it is used for RNA synthesis. We have studied how rabbit dentine calcification is affected by zinc acetate, and zinc concentrations in the liver, cortex renis, submandibular gland and dental pulp. Details are as follows.1) When we injected zinc acetate into a rabbit, we could see a brown line which represented zinc ferricyanide or zinc sulfate. We found it to be zinc by means of electro-micro-analysis. The minimum zinc volume was 1 mg/kg, which made the accumulation line. With up to a 30 mg/kg volume we did not see any effect on calcification. The accumulated volume corresponded to the administered volume.2) Compared with zinc of the chelating type, the ionic type of zinc accumulated more in dentine.3) We assumed that zinc was moved in the blood vessels as a binding substance with serum protein and then accumulated in dentine. Zinc took at least 20 or 30 minutes to accumulate in dentine after intravenous injection.4) Zinc concentrations were significantly increased in dentine pulp.

The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide

Silverman and Kruger (Somatosens. Res. 5(2): 157–175; 1987) reported that sensory nerve fibers of the dental pulp secrete calcitonin gene-related peptide (CGRP). These are localized exactly where secondary or tertiary dentin calcification occurs. Recently we found that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies in vitro. The purpose of this study is to test whether there is a relationship between the effects of different doses of CGRP and bone colony numbers and/or size. Rat CGRP in different dosages (0.4, 4 and 40 μg/ml in BGJb medium) was added daily to 3 million light density (LD) bone marrow white cells which were harvested from adult Sprague-Dawley rats with the Ficoll-Paque density gradient separation method, then seeded onto a previously prepared feeder layer of fibroblasts in Petri dishes. Seven days after adding CGRP, in the controls without CGRP there were 2 bone colonies; with 0.4 μg of CGRP there were 4 colonies; with 4 μg of CGRP there were 6 colonies; with 40 μg there were 9 colonies, indicating there was a significant increase in number of bone colonies with an increase in dose of CGRP between individual groups, respectively ( p<0.0005 and p<0.0001). In another experiment, intravenous injection of 10 μg of rCGRP/kg body weight was performed two hours before surgery. LD bone marrow white cells were collected and seeded onto a feeder layer in Petri dishes exactly as described above. After 10 days, an average of 3 bone colonies per 3 million LD white cells were found in the rCGRP-injected group as compared to 2 colonies in the control group ( p<0.05). The first result indicates that CGRP will increase its osteogenic stimulating effect with an increase in dose. In the second experiment, it was shown that intravenous administration of CGRP will also increase bone colony formation by stimulating either stem cell mitosis or osteoprogenitor cell differentiation.

The rate of incisor dentine calcification and of mandibular growth in the molar region of the ovariectomized rat

In 14 ovariectomized and 15 control rats, killed at intervals over a 117-day period, the height of the mandible in the molar region was determined radiographically. Mandibular height was significantly related to time after operation in both groups, but their rate of mandibular growth was not significantly different. In a separate study, the rate of dentine formation in the right mandibular incisor of 9 ovariectomized and 10 control rats was calculated from the distance between two fluorescent labels injected at known time intervals. The rate of dentine apposition was measured at 117 and 201 days after ovariectomy. In both groups, the distance between the fluorescent labels was significantly correlated with the time interval between the injections. There was no significant difference between the rate of dentine calcification in either group. Transient changes in dentine apposition may have occurred post-ovariectomy which would be undetected at either 117 or 201 days after operation. There was thus no evidence for an effect of ovariectomy on either the rate of mandibular growth or dentine apposition.

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1 alpha,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine.

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.

The dentino-enamel junction: A structural and microanalytical study of early mineralization

The spatial localization of enamel and dentin apatite crystals of the rat tooth has been studied by electron microscopic methods--bright field, selected-area dark field, and electron spectroscopic imaging. The sequential events of dentin calcification followed by the formation and growth of enamel crystals were determined and compared to previous studies. In dentin, initial sites of mineral deposition occur in areas subjacent to the dentino-enamel junction (DEJ). The subsequent expansion of these deposits progresses towards the DEJ to the terminal ends of dentin collagen fibrils. Concomitantly, an electron-dense enamel matrix is released by ameloblasts; with the presence of this matrix, the growth of enamel crystals occurs from the underlying calcified dentin. Enamel crystal growth continues to within close proximity of the plasma membrane of ameloblasts. A close spatial relationship between enamel and the crystals of calcified dentin collagen fibrils was observed by selected-area dark field imaging. Such areas of crystal intimacy show a co-localization of calcium and phosphorus extending from calcified collagen fibrils to enamel sheaths which encase enamel crystals. A working model of the spatial relationship between crystals of dentin and enamel is presented and discussed in light of mechanisms by which calcified dentin may promote the formation of enamel crystals.

Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3′,5′-monophosphate in mouse bone marrow cell cultures

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.

Induction of calcitonin receptors by 1 alpha, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells.

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.

Chapter 9 The ultrastructure and receptor transduction mechanisms of dentine

Publisher Summary This chapter discusses the ultrastructure and receptor transduction mechanisms of dentine. Recent experience has emphasizes that the tubule contents are not fixed rapidly after vascular perfusion, and that sufficient time must be allowed for diffusion of fixative into the tubules. In experiments designed to detect horse-radish peroxidase (HRP) transported to the teeth in nerves, the chapter perfuses the animals in the normal way then washed out the fixative after half an hour to preserve the HRP. The pulps were well fixed, including the cornua, but not the contents of the dentinal tubules. The outer diameters of both this annulus and the peritubular dentine became smaller as the diameter of the lumen of the tubule decreased in the outer dentine. Also, in the outer dentine, both diameters tended to be smaller than those of the tubules in the calcified dentine adjacent to the pre-dentine. In addition, the chapter also investigates the effects of different methods of fixation because there are always uncertainties about whether the lack of cellular material in outer dentine might be because of inadequate preservation.