A surgical technique for administering laser irradiation to rat incisor dentin.

With the advent of lasers in the field of dentistry, various methods have been used to expose dentin to laser irradiation. This was done indirectly through the alveolar bone, as well as directly on dentin wafers and on extracted human teeth. However, dentin is a vital tissue and the only way to show the ramifications of laser irradiation is by directly exposing the dentin to this energy source under in vivo conditions. In the present study we describe a highly reproducible method for accomplishing this. Male Sprague Dawley rats were anesthetized and an incision was made at the junction of the attached palatal mucosa and the unattached buccal mucosa parallel to the maxillary bones on both sides. This procedure exposed the underlying bone where a 2 mm square window, at a point 3 mm caudal and parallel to the gingival attachment, was cut out to the depth of the periodontal ligament. The site was flushed with a 0.9% sterile sodium chloride solution and gently dried with gauze. The beam of the laser was focused in the middle of the 2 mm square window and a pre-determined energy level and pulse duration (2 W 50 ms or 10 W 50 ms) were selected. The focal length guide was held at right angles to the dentin surface and in contact with the maxilla at all times. The energy densities of either 52.6 or 263.2 J/cm(2) were delivered to the 'root' dentin by a single pulse. The survival rate was 92%. The animals that died did so while under anesthesia and not after exposure to laser irradiation. The animals that survived tolerated the surgery and the laser irradition very well, regaining (and eventually surpassing) their initial weight 3 days after the procedures were performed. The thickness of the dentin that was exposed to laser irradiation was not completely penetrated by the energy levels used. At both energy levels some dentinal tubules appeared blocked and evidence of melted dentin was apparent. However, at the higher energy level two distinct zones were observed in the funnel-shaped lesion. In the inner zone distinct dentinal tubules were not readily visible, while in the outer zone the tubules were opened and completely free of debris. This technique is highly reproducible and yields consistent results with little or no stress to the animals. Although the energy levels used did not penetrate the entire thickness of the dentin, the depth of penetration of the laser beam appears to be related to the energy level used and the thickness of the dentin layer present.

Effect of adriamycin on the RPC C2A cloned cell line from rat incisor pulp.

BACKGROUND AND METHODS: RPC C2A cells, cloned from Wistar rat incisor pulp, were grown in culture 2-7 days after exposure to adriamycin at a concentration of 0.1 mg/L for 2 hours. Morphological changes, labelled proline incorporation and alkaline phosphatase activity were studied in response to this treatment. RESULTS: At the light microscopic level, colonies of enlarged cells appeared 2 days after adriamycin treatment. The size of these colonies increased during the course of this study. The control samples showed no apparent changes. At the electron microscopic level, the small cells in the adriamycin-treated cultures showed fewer vesicles than the controls, but more prominent rough endoplasmic reticulum. However, the enlarged cells contained an abundance of vesicles, and few profiles of rough endoplasmic reticulum. Radiolabelled proline incorporation in the control group was significantly higher than the experimental after 2 days in culture, but showed no significant difference after 7 days. Histological staining for alkaline phosphatase showed that there was a slightly higher intensity in the control samples than in the experimental after 7 days in culture. Quantitative analysis showed that there were more alkaline phosphatase stained cells in the control than in the experimental cultures at this time. CONCLUSIONS: This study has shown that the RPC C2A cells will respond to adriamycin treatment in vitro, where the acute effects were quite different from the protracted effects with respect to labelled proline incorporation and alkaline phosphatase activity.

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.

The effects of adriamycin on dental proteins formation and mineralization in vitro.

Second maxillary molars of 4-5 days old golden hamsters were exposed for 2 h in vitro to 1 mg/L adriamycin, rinsed and subsequently cultured up to 7 days without the drug. At days 3, 5 or 7 of culture the synthesis of extracellular tooth matrices and their mineralization were examined by measuring the incorporation of 3H-proline and the uptake of 45Ca and 32PO4 by the explants during a 24 h pulse labeling. Compared with unexposed control explants, exposure to adriamycin for the first 2 h of culture had no effect on total biosynthesis of proline-containing matrix proteins. However, at days 3 and 5 of culture it increased the quantity of water-soluble enamel matrix proteins (amelogenins). Adriamycin also strongly reduced the amount of organically-bound 32P-activity in a fraction extractable with guanidine-HC1-EDTA only, allegedly containing a mixture of mineral-associated proteins from both enamel and dentin. Since this decrease of 32P-activity coincided with the formation of osteodentin in the pulp as shown previously in histological and electron microscopical studies, it was speculated that osteodentin matrix may not contain the highly phosphorylated, dentin-specific phosphoproteins (DPP). Adriamycin also affected the uptake of 45Ca and 32PO4. At day 3 these values were slightly higher than control values but lower at days 5 and 7. It therefore appears that a 2 h exposure to adriamycin in concentrations as low as 1 mg/L causes an acceleration of secretory amelogenesis by tooth germs in vitro. It also induces pulp cells to form osteodentin.

The effect of adriamycin on dentinogenesis and 3H-thymidine incorporation into the enamel organ of the rat incisor.

The effect of adriamycin (5 mg/kg) on 3H-thymidine incorporation and on dentin formation was studied in rat incisors. Male Sprague Dawley rats received an intravenous injection of adriamycin. Some of these also received a subcutaneous injection of 3H-thymidine at a dose of 2 mCi/kg one day later. One group of control animals received an intravenous injection of a volume of physiological saline equal to that of the adriamycin dose. Another group received physiological saline, and one hour later was given an additional injection of 3H-thymidine at a similar dose as above. All the animals were killed by perfusion with 2.5% phosphate buffered glutaraldehyde 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 32 d after 3H-thymidine treatment. Light microscopy revealed irregular dentin deposits between the mantle and circumpulpal layer of the labial dentin at 16 d. Within these deposits were trapped cells. The latter, through radioautographic labelling, appeared to be cells from the odontoblast layer. Also, the labelling pattern of the enamel organ in both the control and experimental groups indicated that the eruption rate of the tooth was not affected. Serial sectioning and examination of the lingual portion of the incisors at 28 d revealed a lack of dentin formation and a failure in the closure of the apical foramen. Electron microscopic observations showed an irregular and random arrangement of collagen fibers within the deposits of irregular dentin, and the presence of twisted odontoblastic processes. Examination of the lingual surface showed the presence of fibroblasts and collagen fibers bridging the gap that resulted from the failure in dentin formation. These cells, which were similar to periodontal ligament cells, appeared to have arisen from that area. These results indicate that adriamycin has no effect on tooth eruption, but has a reversible effect on the function of secretory odontoblasts, which manifested itself as a periodic deposition of irregular dentin on the labial surface, and on dentin formation on the lingual surface which manifested itself as a failure in dentin formation, and consequently, the closure of the apical foramen.

Effect of adriamycin on hamster molar tooth development in vitro: 1. Morphological changes.

The effect of adriamycin (1 mg/liter) on the development of the golden hamster 3-day-old second maxillary molars (M2) was investigated in vitro. Exposure of the molars to 1 mg/liter adriamycin during the first 2 hours of culture produced smaller teeth 3-7 days later, as determined by measurements of dry weights and by histological observations. Higher doses caused severe necrosis. The more differentiated pulp fibroblasts showed osteodentin formation 3 days after treatment with adriamycin (1 mg/liter), while the more immature ones underwent necrosis. The phenotypic changes brought on by the drug were permanent, and osteodentin continued to be formed throughout the course of this study. In addition the cervical loop region was inhibited from growing, while the production of the matrices of enamel and dentin appeared to be increased at 3 and 5 days after treatment. Electron microscopy of the forming osteodentin matrix revealed a random arrangement of banded collagen fibers during the early stage of osteodentin formation. As more matrix was formed, the collagen became quite compact and appeared quite similar to dentin. Finally, matrix vesicles were found among the collagenous matrix that was not yet mineralized. With the exception of the increased production of enamel and dentin, these in vitro results confirmed those earlier in vivo studies on the effect of adriamycin on rat incisor tooth.

Osteodentin formation in rat incisor as visualized by radioautography after 3H-proline administration.

Osteodentin formation was studied in rat incisor pulp after adriamycin administration. Male Sprague Dawley rats (100 +/- 5 gm) were injected intravenously with adriamycin (5 mg/kg body weight), and after 7 days they were again injected intravenously with 3H-proline (3 microCi/gm). These animals were killed in groups of three from 5 minutes to 4 hours after proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by 2.5% phosphate-buffered glutaraldehyde. Control animals injected with only physiological saline, and 7 days later with 3H-proline (3 microCi/gm), and were killed at the same time intervals. Radioautography on sections showing osteodentin formation revealed that at 5 minutes after 3H-proline injection the labeling was located over the cells associated with the osteodentin matrix. At 1 hour after injection the labeling was located over the cells and the matrix, while at 4 hours the labeling was seen only over the matrix. It therefore appears that at least a proline-containing component of the osteodentin matrix is synthesized and secreted by the cells associated with it.

The effect of streptozotocin on the secretory activity of ameloblasts in rat incisor as revealed by radioautography after 3H-proline administration.

The effect of a diabetogenic dose of streptozotocin on the secretory activity of ameloblasts was investigated in the rat incisor by radioautography. One group of male Sprague-Dawley rats was injected intravenously with streptozotocin in citrate buffer (pH 4.5). One hour later, this group was again injected intravenously with 3H-proline (2 mCi/kg). A control group of animals was injected with 3H-proline only. All the animals were sacrificed in groups of three at 5 min, 1 h, 2 h, 4 h and 8 h after 3H-proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by an additional perfusion with 2.5% phosphate-buffered glutaraldehyde. The incisors were extracted with the jaws, demineralized, and prepared for radioautographic observations and analysis. The principal effects of streptozotocin were as follows: There was an inhibition of 3H-proline incorporation into the secretory ameloblasts at 5 min after injection. This was followed by a larger uptake and a slower passage of the label out of the cells into the enamel matrix than that seen in the control sample. Finally, there was a slower secretion of labeled proteins out of Tomes' processes between 1 and 4 h after injection. Therefore, streptozotocin had a temporary inhibitory effect on the incorporation and secretion of 3H-proline by the secretory ameloblasts of the rat incisor. This effect was present for about 4 h and was completely reversed 9 h after streptozotocin injection.

The initiation of osteodentin formation in the rat incisor after adriamycin administration.

In order to study the initiation of osteodentin formation in rat incisors, animals were injected intravenously with adriamycin (5 mg/kg body weight), and killed from 2 to 7 days after injection by perfusion with a 2.5% buffered glutaraldehyde solution. Control animals, injected with only physiological saline, were treated in the same manner. Three days after adriamycin injection aggregations of mesenchymal cells were observed along the mesial and lateral walls of the pulp chamber. Between 3 days and 7 days osteodentin production was observed at the sites where the mesenchymal aggregations were previously observed. Electron microscopic observations revealed that the cells involved in the aggregates were larger and contained more profiles of rough endoplasmic reticulum and secretion granules than the unaffected pulp cells. The osteodentin matrix first appeared as a scant deposition of collagen fibers between the cells. As more collagen fibers were deposited the matrix became much denser. Some cells that initially formed the mesenchymal aggregates were completely enclosed by the increased deposition of the matrix. It therefore appears that osteodentin formation, as observed in the rat incisor pulp after adriamycin administration, is the result of an abnormal differentiation of pulp mesenchymal cells.

The extent of the necrotic lesion in the apical end of the rat incisor pulp seen after Adriamycin administration.

The extent of the necrotic lesion produced by adriamycin in the apical end of the rat incisor was investigated. Male Sprague Dawley rats were injected intravenously with adriamycin (5 mg/kg body weight), and sacrificed by perfusion with a 2.5% buffered glutaraldehyde solution 1 d after injection. Controls injected with only physiological saline were treated in the same manner. One micron thick plastic serial sections were prepared from the apical end of the incisor, and a schematic representation of the entire lesion as seen in longitudinal section was reproduced from each fiftieth section. Cell destruction was observed for 2.2 mm within the pulp. The lesion extended further incisally in the lingual portion than in the labial portion. The bulbous portion of the odontogenic organ was not affected. This study indicates that the cytotoxicity of adriamycin appears to affect mainly proliferating immature precursor cells within the mesenchyma, and the preodontoblasts.

The effect of adriamycin on rat incisor one day after administration.

The effect of adriamycin on rat incisor was investigated 1 day after administration. Rats were injected intravenously with adriamycin at a dose of 5 mg/kg body weight and sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. The principal effect of the drug on the incisor was the production of cell degeneration. This was extensive in the apical region, being present along the entire periphery of the dental papilla. In sections examined more incisally, cell degeneration gradually disappeared from the labial portion of the incisor but was present in the lingual portion. This degeneration of cells was not present at the site where mature odontoblasts had differentiated on the lingual surface of the pulp chamber. It appears that the affected cells were early preodontoblasts and the precursors of preodontoblasts. However, as these cells became more differentiated they apparently became more resistant to the drug's effect.

A light and electron microscopic study of osteodentin formation in the rat incisor after adriamycin administration.

The effect of adriamycin (10 mg/kg body weight) on the rat incisor was investigated in 8-day-old animals at 9 days and 14 days after subcutaneous injection. The drug produced changes that were still present 14 days after administration. During this time osteodentin formation, which appeared to be the principal effect of the drug on the incisors, occurred to such an extent that in some regions of the teeth the pulp chamber was almost completely occluded. The formation of osteodentin began at the periphery of the pulp and gradually advanced towards the central region. Moreover, in some sections of the incisor the dentin layer was greatly reduced to a thin superficial layer, while osteodentin surrounded most of the pulp chamber. Cells that appeared to be differentiated pulp-mesenchymal cells were found within as well as on the surface of the irregular osteodentin matrix. Since this drug has been used in the treatment of childhood osteosarcomas, the possibility of dental abnormalities developing in these children cannot be overlooked.

An ultrastructural study of the effect of streptozotocin on the secretory ameloblasts of the rat incisor.

The acute effect of a single injection of streptozotocin (75 mg/kg) on the secretory ameloblasts of rat incisor was found to be reversible after 4 h. This effect was manifested ultrastructurally by an accumulation of secretion granules within the Golgi apparatus and a diminished accumulation of secretion granules within Tomes' process, thus suggesting a temporary inhibition of secretion. The long term effect of streptozotocin was characterized mainly by large accumulations of secretion granules within the Golgi apparatus, the presence of many lysosomal structures, an abnormal redistribution of secretion granules, and the presence of large amounts of electron-dense material within the intercellular spaces. Because the electron-dense material resembled the enamel matrix, this, and the above changes, suggested that there was a pronounced inhibition in the movement of secretion granules into Tomes' process, as well as an ectopic secretion of the enamel matrix protein.