Kojiro Kurisu

Effects of epidermal growth factor on osteoblastic cellsin vitro

SummaryThe effect of epidermal growth factor (EGF) on clone MC3T3-El cells that have osteoblastic activity was examined by phase-contrast microscopy and electron microscopy; hydroxyproline content, collagen synthesis, collagen pattern, and alkaline phosphatase (ALP) activity were also determined. We found that EGF (0.4 ng/ml) transformed the cells from their normal polygonal shape to a spindle-like morphology by 8 h. This hormone also caused dose-related suppression of hydroxyproline content and ALP activity which was detectable 2 days and 1 day, respectively, after EGF addition. Indomethacin did not affect hydroxyproline content and ALP activity, suggesting that the effect of EGF on the cells may not be mediated by prostaglandins. Epidermal growth factor at concentrations of 2 to 50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was stimulated. Electron microscopy demonstrated that collagen fiber formation was also reduced by EGF; an immature type of fibril was observed compared with the typical cross-striated one in the controls. Moreover, the hormone treatment also resulted in the appearance of type III collagen in addition to the type I already present in the cells. These suppressive effects of EGF on MC3T3-El cellsin vitro suggest that this hormone may be involved in bone remodellingin vivo as well.

Immunofluorescence localization of type I and type III collagen and fibronectin in mouse dental tissues in late development and during molar eruption

Affinity-purified antibodies produced intense staining for type I collagen in alveolar bone matrix and predentine, and moderate staining in the dentine matrix, lamina propria, connective tissue invaginating into papillary layer of the enamel organ, dental sac and periodontal ligament. No staining occurred in oral epithelium, stellate reticulum, stratum intermedium, ameloblasts and odontoblasts. Fibronectin was distributed similarly except at the interface between the epithelial diaphragm and pre-odontoblasts where type I collagen was absent but fibronectin was present. In contrast, type III collagen showed strong staining in the periodontal ligament and lamina propria but no staining in bone matrix, predentine, dentine and at the interface between the epithelial diaphragm and pre-odontoblasts. The staining pattern for type III collagen was similar to that of type I and fibronectin in other tissues including endosteal reticular tissue, the connective tissue invaginating into papillary layer and the extracellular matrix of the pulp.

Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine

Rat bone marrow cultures containing 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.

Distribution of type I and type III collagen in the developing periodontal ligament of mice.

In order to localize type I and type III collagen in developing periodontal ligament, immunofluorescent and immunoelectron microscopic examinations were undertaken. The materials used were the maxillary molars of CF1 mice, 13 days to 6 months old. The antibodies used were monospecific polyclonal antibodies against type I or type III collagen raised in rabbits or guinea pigs, respectively. Single- and double-staining methods were employed in immunofluorescent as well as immunoelectron microscopic examinations. In immunofluorescent examinations, during the development of the periodontal ligament, periodontal fibers showed intense and homogenous staining for both type I and type III collagen; while Sharpeys fibers in the alveolar bone showed a heterogenous staining in cross and longitudinal sections for both types of collagen. In immunoelectron microscopic examinations, fibrils of periodontal fibers showed positive staining for type I and type III collagen simultaneously and had characteristic cross-banding and had a large diameter (ranging 40 approximately 80 nm) which remained constant during development. Sharpeys fibers in the alveolar bone or in the cementum showed the same staining pattern as the periodontal fibers, except for an afibrillar area which showed negative staining for both type I and type III collagen. The results obtained suggest that periodontal fibers and Sharpeys fibers consist of cofibrils of at least type I and type III collagen.

Primary and secondary culture of rat ameloblasts in serum-free medium

SummaryEnamel is the hardest tissue in vertebrates. Ameloblasts are derived from epithelial cells and are responsible for enamel formation. They secrecte enamel matrix components in which amelogenins are the major proteins, the biochemical properties of which are well known. However, little is known about the characteristics of ameloblasts themselves or about the functions of amelogenins. In this study, we developed a novel primary and secondary culture system for ameloblasts using a monoclonal antibody which recognized amelogenin (En3). The cell layer on dentine removed from rat mandibular incisors was isolated and cultured in low calcium, serum-free medium. Primary culture was performed on collagen-coated culture plates and typically, two types of cells appeared. One major type changed morphology after the addition of a high concentration of calcium to the medium. Expression of amelogenin was shown as cytoplasmic particles in these cells using En3. In the secondary culture, expression of amelogenins was also observed. In this system, the cells grew and maintained the expression of amelogenin for about 3 weeks.

Recombinant LD78 protein, a member of the small cytokine family, enhances osteoclast differentiation in rat bone marrow culture system

Cytokine LD78 is a member of the small inducible protein family involved in cell growth, wound healing and inflammation. However, its exact function is not known. In this study, we demonstrated that recombinant LD78 alpha and its variant LD78 beta proteins stimulate osteoclast-like cell formation in rat bone marrow cultures in the presence of 1 alpha, 25-dihydroxyvitamin D3. This enhancing activity was independent of prostaglandin synthesis. This is the first report which describes the effect of proteins involved in the small inducible protein family on the differentiation of osteoclasts.

Demonstration of type III collagen in the dentin of mice

It has been reported that, although type III collagen is present in human dentin where there is dentinogenesis imperfecta and in reparative dentin, it is absent in normal dentin. In a preliminary study, however, we observed evidence showing that small amounts of fibers showing positive labeling for type III collagen are present in the molars of normal mice. In the present study, in order to localize type III in normal dentin, immunofluorescent and immunoelectron microscopic examinations of the molars of normal mice were carried out using affinity-purified antibodies to mouse type III and type I collagen. The fibers positive for type III collagen were much more frequently observed in the root than in the crown. These fibers ran in peritubular dentin or near that in parallel to them. The incidence of the existence of dentinal tubules associated with type III collagen-positive fibrils either in or near peritubular dentin was low. These fibrils positive for type III collagen showed a clear cross-banding. In dentinal tubules, unusual collagen aggregations, segment long-spacing-like and fibrous long-spacing-like structures which were intensively stained for type I collagen but weakly so for type III collagen were seldom observed. Type III collagen-positive fibers often extended towards the pulp beyond the odontoblast layer, suggesting that these fibers were produced, at least partly, by the pulp cells.

Effects of retinoic acid on receptors for epidermal growth factor in mouse palatal mesenchymal cells in vitro

Retinoic acid (RA), one of the inducers of cleft palate in vivo, specifically increased the 125I-epidermal growth factor (EGF) binding capacity of embryonic palatal mesenchymal cells; the increase was completely inhibited by actinomycin D, suggesting that transcription is required for this change. When the increase in EGF receptors was related to the stimulation of DNA synthesis by EGF in the presence of various concentrations of RA, the maximum stimulation of [3H]-thymidine by EGF was at 10(-9) M RA, but the maximum increase in the number of EGF receptors was at 10(-6) M RA. Thus the increase in the number of EGF receptors cannot be related to EGF-stimulated DNA synthesis.

Immunohistochemical study of the relationship between extracellular matrix and root bifurcation in the mouse molar

In order to investigate epithelial-mesenchymal interaction during root bifurcation, the distribution of type III and I collagen, fibronectin and laminin in the epithelial-mesenchymal junction between the dental epithelia (epithelial diaphragm and interradicular process) and the cells of the dental papilla (or pre-odontoblasts) was examined, using maxillary first molar tooth germs of CF1 mice from day 1-16 after birth. Three-dimensional reconstructions of the immunofluorescent patterns were made from serial sections of tooth germs from day 3-9, stained with the antibodies against the collagens. The findings were as follows. (1) Type III collagen was first seen in the epithelial-mesenchymal junction at the tip of the interradicular process, where it sprouted from the epithelial diaphragm, and spread along the interradicular process toward its base, accompanied its extension, and then disappeared on completion of root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during and after root bifurcation. (2) Type I collagen appeared in the epithelial-mesenchymal junction at the base of the interradicular process, where it sprouted from the epithelial diaphragm and spread toward the tip of the interradicular process, following its extension, and increased on completion of the root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during or after root bifurcation. (3) Fibronectin and laminin remained constant in the epithelial-mesenchymal junction, both at the interradicular process and the epithelial diaphragm, during and after root bifurcation. These findings suggest that type III collagen may play a significant role in the early stage of root bifurcation in the molar.

Immunocytochemical Demonstration of Simultaneous Synthesis of Types I, III and V Collagen and Fibronectin in Mouse Embryonic Palatal Mesenchymal Cells In Vitro

An immunocytochemical study was made on the palatal mesenchymal cells obtained from mouse embryos during palatal development with special reference to synthesis of types of collagen and fibronectin in vitro. Most cells showed positive staining with antibodies against the four proteins examined. The staining for type I collagen was most intense among the four proteins and was distributed perinuclearly. The staining for type III collagen was quite similar as that for type I collagen but less intense, whereas that for type V collagen was weak and its staining pattern was different from those for types I and III collagen in that the surface of the plasma membrane, in addition to the perinuclear cytoplasm, showed weak staining for type V collagen. Antibodies to fibronectin showed perinuclear and extracellular fibrous staining. These data suggest that palatal mesenchymal cells synthesize types I, III, and V collagen and fibronectin simultaneously.