Proceedings of the Finnish Dental Society. Suomen Hammaslaakariseuran toimituksia最新文献

The use of procedures adapted from a routinely successful method of culturing bovine bone has led to the first system for the study of dentinogenesis in vitro. Two types of cells have been grown from pulp obtained from the growing root tips of impacted third molars extracted from 14- to 19-years olds: (1) epithelial-like cells that are probably derived from fragments of the epithelial root sheath and (2) odontoblast-like cells. The cultured epithelial-like cells grow out in distinctive rounded plaques while the odontoblast-like cells are tethered to and/or grow on top of the epithelial-like cells. The odontoblast-like cells produce mineralized matrix by 10 days when cultured on a defined mineralization formula containing conditioned medium obtained from fetal bovine bone cell cultures. Growth factors in this conditioned medium are important to cell proliferation and growth and to the synthesis of mineralized matrix. Sequential enzyme digestion in dispase and dispase/collagenase in serum-free Dulbecco's Modified Eagle's Medium is essential to obtaining adequate cell yields from the apical 3-5 mm of the developing root. Reduction of the number of fibroblasts by treating cultures with dispase in Tyrode's solution midway through the initial growth period enhances the purity of these cell cultures.

CO2 laser may be beneficially applied to teeth in preventive and restorative dentistry, but its physiopathologic effects on the dental pulp have not been reported. To address the dynamic pulpal changes induced by CO2 laser the purpose of this study was to examine such changes in pulpal microcirculation. The enamel surfaces of 4 cat canines were irradiated by CO2 laser beam, delivered by either a handpiece or microslad with a focal spot diameter of 0.21mm and 0.33mm, respectively. Exposure powers of 2.6-20 W were used for durations of 0.05-0.2 sec, resulting in energy density range of 304-1440 J/cm2. Non-invasive measurement of pulpal blood flow (PBF) by a laser Doppler flowmeter was maintained continuously before and following CO2 laser irradiation. CO2 laser irradiation resulted in an immediate increase in PBF, which subsided after 2-3 min. Large pulps responded with a significantly higher PBF increase than small pulps. These results confirm that CO2 laser irradiation of the tooth surface has a thermal effect on the pulp, which is moderated by the insulating capability of dentin. It appears that the associated thermal damage is not extensive when laser exposure parameters are such as used herein.

We investigated whether the expressed phenotype of osteoblasts and odontoblasts is changing with increasing maturity of the cells. Thus we determined, using in situ hybridization techniques, whether the expression of mRNA's for osteocalcin and the alpha 1 type 1 collagen chain was different in newly developed and more mature cuboidal osteoblasts of the primary and secondary spongiosa of radii of 8 day old rats, in mature cuboidal and older flat osteoblasts in the metatarsals of pig embryos, and in apical and coronal odontoblasts of the developing unerupted molars of pig embryos. The results indicate that newly differentiated osteoblasts in the primary spongiosa of the 8 day rat radius contained approximately the same amount of type 1 collagen message as more mature osteoblasts in the secondary spongiosa. Osteocalcin mRNA, on the other hand, was undetectable in the newly differentiated osteoblasts but clearly detectable in the mature osteoblasts of the secondary spongiosa. When we compared expression of osteocalcin and collagen type 1 mRNA in mature cuboidal and older flat osteoblasts, we found that the amount of osteocalcin mRNA relative to collagen type 1 mRNA was higher in flat osteoblasts than in cuboidal osteoblasts. In odontoblasts, however, the steady state level of collagen type 1 mRNA was higher in the older coronal odontoblasts, and the level of osteocalcin message lower, when compared to the younger apical odontoblasts. The results indicate that relative levels of osteocalcin and collagen mRNA in osteoblasts and odontoblasts vary depending on the stage in their secretory lifetime. This heterogeneity of the osteoblast and odontoblast population suggests that the composition of the matrix produced by these cells also differs.

In developing teeth the differentiation of odontoblasts is triggered by the enamel epithelium and is tightly coupled with morphogenesis. There is substantial evidence that even in mature teeth the cells of the dental pulp retain the capability to differentiate into odontoblasts under suitable conditions. However, cells from other than the dental mesenchymal cell lineage apparently do not possess this potential. Thus, it is conceivable that the dental mesenchymal cells acquire cell type-specific potential to differentiate into odontoblasts during their developmental history. Therefore, the understanding of the mechanisms which regulate the terminal differentiation of odontoblasts requires that the molecular changes and mechanisms that are associated with their progressive determination be clarified. It can be speculated that there are key transition points in the developmental sequence during which the mesenchymal cells acquire new levels of differentiation. These include, (1) the condensation of the neural crest-derived mesenchymal cells around the epithelial bud, (2) their entrance into the dental papilla lineage during cap stage, and (3) the differentiation of the cells underlying the enamel epithelium into odontoblasts during bell stage. The transition points are conceivably characterized by amplification or onset of expression of new sets of genes encoding transcription factors, growth factors as well as structural proteins. We have applied in situ hybridization for localization of the expression of two growth factors during mouse molar morphogenesis: transforming growth factor beta 1 (TGF beta 1) and int-2 (a proto-oncogene coding for a fibroblast growth factor-related protein). During bud stage, expression of TGF beta 1 was first detected in the epithelium and shortly thereafter in the condensed dental mesenchyme. The expression was weak during early bell stage but a high number of transcripts appeared in secretory odontoblasts as well as in presecretory ameloblasts. int-2 mRNA appeared in the dental papilla mesenchyme at the onset of cap stage, persisted in the cuspal mesenchyme during bell stage and was lost upon completion of morphogenesis. Our findings suggest that cell type-specific expression of TGF beta 1 and int-2 is associated with phenotypic properties of the odontoblastic cell lineage. For instance, TGF beta 1 may regulate matrix deposition by increasing tenascin and syndecan expression in the condensed dental mesenchyme and by controlling dentin matrix deposition by odontoblasts. TGF beta 1 and int-2 may also be involved in signalling between the epithelial and mesenchymal tissues and in regulation of gene expression at the transition points of the developmental sequence that leads to the differentiation of odontoblasts.

Several extracellular matrix (ECM) proteins have been isolated from dentin and shown to be synthesized by odontoblasts. Highly phosphorylated proteins (HP), the phosphophoryns, are specifically found in dentin and are synthesized only by odontoblasts. Phosphophoryns are probably involved in controlling the site and/or the rate of circumpulpal mineralization. Other moderately phosphorylated (MP) and low phosphorylated (LP) proteins have also been detected in dentin, but have not been studied extensively. A 53 kDal dentin sialoprotein (DSP) that resembles bone sialoproteins is expressed by odontoblasts and by pulp cells, but essentially by no other cell type. The function of this protein is unknown. Osteocalcin (bone Gla protein) is synthesized by osteoblasts and by odontoblasts and found in the ECM of bone and dentin. Other tissues or cells (including pulp) do not express osteocalcin. The highly specific nature of these dentin and bone ECM proteins suggests that antibody and molecular probes for the phosphoproteins, DSP and osteocalcin could be valuable in studies on the differentiation and identification of odontoblasts and their precursors. Other bone NCPs shown to be present in dentin may also prove to be important in dentinogenesis.

Recent studies suggest that TGF-beta 1 controls events in embryogenesis via its modulation of the extracellular matrix (ECM). During tooth development, histomorphogenetic and cytodifferentiation events are regulated by ECM-mediated epithelial-mesenchymal interactions. In this study, we have investigated the in vivo relationship of the localization of TGF-beta 1 to various ECM proteins, including glycosaminoglycans (GAGs), fibronectin, collagens I and III and to the enzyme alkaline phosphatase, at the early and late bell stages of rat molar tooth development. We have used polyclonal antibodies specific to peptides present in the precursor and processed forms of TGF-beta 1, fibronectin and Collagens I and III and immunohistochemical methods of detection. GAGs were stained with Alcian Blue GX and alkaline phosphatase was detected with the enzyme Naphthol-AS B1. Our results indicate that both the intracellular and extracellular forms of TGF-beta 1 are expressed in a site-specific manner, and that the growth factor colocalizes with alkaline phosphatase, fibronectin, collagens I and III and GAGs during the bell stages of tooth development. These data support the hypothesis that in tooth development, the actions of TGF-beta 1 may contribute to important qualitative changes in the composition and structure of extracellular matrices.

The mechanism of bone destruction in periapical lesions was studied using a rat model system. Periapical lesions were induced by pulp exposure and infection from the oral environment. Lesions expanded most rapidly between induction on day 0 and day 15 ("active phase"), with enlargement occurring at a slower rate thereafter (days 20 and 30, "chronic phase"), as assessed by radiography and automated image analysis. Pooled extracts of day 15 periapical tissues contained significant levels of bone resorbing activity (BRA), as determined by 45Ca release from fetal rat long bones. Normal rat dental pulp and periodontal ligament contained no activity. In kinetic experiments, highest levels of BRA were detected in active phase tissues on days 10 and 15, with BRA declining thereafter in chronic phase tissues to near baseline levels by day 30. In characterization studies, BRA in pooled day 15 tissue extracts was unaffected by treatment with polymyxin B, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating an active moiety distinct from bacterial LPS, probably a protein(s). FPLC gel filtration chromatography revealed that BRA could be resolved into four major peaks, of MW 30-60 kD (Peak I); 15-20 kD (II); 1-2 kD (III); less than 1kD (IV), consistent with the presence of the following bone resorptive mediators: (I) cytokines TNF alpha, TNF beta and/or unprocessed IL-1 alpha; (II) processed IL-1 alpha and/or IL-1 beta; (IV) PGE2. These findings demonstrate that bone resorbing activity is temporally related to bone destruction, and that the activity present during the rapid phase of periapical lesion expansion is primarily attributable to bone resorptive cytokines.

Aging of cat tooth pulp nerves involves ultrastructural changes, and changes in the expression of some neuropeptides and in the expression of the receptor for nerve growth factor. Electron microscopy of old pulps demonstrates loss and degeneration of unmyelinated and myelinated axons, as well as demyelination. Immunohistochemical findings show a marked age-related decrease in pulpal calcitonin-gene related peptide- and substance P-like immunoreactivity, and a reduction in nerve growth factor receptor-like immunoreactivity. Changes in neuropeptide expression are not entirely due to loss of nerve fibers, since most aging pulps contain nerve growth factor receptor-positive fibers which lack neuropeptide-like immunoreactivity. The changes reported here parallel the observation that there is an age-related reduction in sensitivity to pulpal stimulation, but may also contribute to the development of oral sensory phenomena such as neuropathic pain. Moreover, the senescent transformation of pulpal nerves probably affects hemoregulation of the pulp, and may thus have consequences for pulpal extraneuronal tissue.

There is a general agreement that the extracellular environment plays a critical role in controlling cell behavior. Thus, significant research efforts have focused on understanding the effects of extracellular matrix proteins on cell function. In particular we have focused on determining the role of adhesion proteins in the regulation of root formation. Using an OPN antibody, 2arN (generously provided by Drs Craig and Denhardt), the expression of OPN during root formation was determined. OPN (osteopontin) is a bone-associated adhesion protein. OPN was expressed in the dental follicle region of molars obtained from 3 day old CD-1 mice, but was not expressed in the odontoblast layer. In contrast by day 8, positive staining was noted in the odontoblast layer, as well as in the area of Hertwig's epithelial root sheath. However, at this same time point no positive labeling for 2arN was observed in the enamel organ or in the dental papillae cells. By day 15 positive staining for OPN was seen in the area of the periodontal ligament, as well as the region of primary deposition of extracellular matrix onto dentin. Also determined was the ability of fibronectin, OPN and dentin sialoprotein (DSP) to promote the attachment of dental ectomesenchymal cells, in vitro. Interestingly, these cells attached remarkable well on bacteriological dishes (control) in the absence of an adhesion protein. DSP did not increase cell attachment beyond that observed for control cells. In contrast, both fibronectin and OPN enhanced cell attachment. These studies, while preliminary indicate that OPN is expressed in a unique fashion during root development, thus suggesting a regulatory role for such adhesion proteins during root formation.

Changes in pulpal vessels in experimentally induced acute and chronic pulpitis in dog tooth were investigated using corrosive resin casts and scanning electron microscopic examination. Following a cavity preparation without water spray, increased permeability of blood vessels occurred in the primary stage of acute pulpitis. This was evidenced by the extravasation of resin from the vessel. This phenomenon was found initially in the venular network as well as in the capillary network located under the dentin. The morphological change was minimal in the vascular network underneath the cavity. This is in contrast to an expanded and tortuous vascular network representing an ulceration which was found around an abscess in chronic pulpitis. Furthermore, formation of vascular loops and AVAlc close to the inflamed region may represent a protective change in the pulp against inflammation.