Yasuyoshi Ohsaki

Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA‐treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase‐polymerase chain reaction revealed that the CsA‐treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA‐treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% ± 2.7% and 24.3% ± 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% ± 5.3% and 81.3% ± 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen‐coated latex beads. Fibroblasts isolated from CsA‐treated gingiva on day 8 and day 30 contained 5.7% ± 0.6% and 9.9% ± 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% ± 5.5% and 33.3% ± 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA‐treated rat gingiva. J. Cell. Physiol. 182:351–358, 2000.

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.

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.

Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development

In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) in tooth development, we treated tooth germ explants of mouse molars with antisense phosphorothioate-oligodeoxynucleotide (ODN) against PTHrP. Antisense ODN-treatment of the explants resulted in the invasion of the tooth germs by bone. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the tooth germs in antisense ODN-treated explants was much lower than that of the control explants. Electron microscopic examination suggested that the antisense ODN-treatment inhibited differentiation of osteoclasts. Treatment of the explants with bisphosphonate or vitamin K2, inhibitors of the differentiation of osteoclasts, induced the invasion by bone into the tooth germs as observed in the antisense ODN-treated explants. The results obtained suggest that PTHrP is involved in the mechanism protecting tooth germs from bone invasion by promoting the differentiation of osteoclasts around them.

Immunohistochemical Localization of Osteopontin in Human Pulp Stones

The organic matrix component of human pulp stones was investigated by immunohistochemistry. Two pulp stones were extracted from the upper molar teeth of two patients suffering from irreversible pulpitis. Both were formed in the center of the pulp cavity and located apart from the dentin walls. After demineralization, serial sections of the stones were prepared and subjected to immunohistochemical procedures using specific antibodies to type I collagen and noncollagenous proteins (osteopontin, osteonectin, and osteocalcin), which are reported to be involved in calcified matrix formation. Type I collagen was localized evenly in the stones, indicating that it is a major matrix component of pulp stones. Strong immunostaining of osteopontin appeared in the peripheral area of the stones, whereas osteonectin and osteocalcin were not detected. We previously reported that dental pulp cells produced osteopontin in vitro. Osteopontin has been commonly found in other pathological calcification, such as urinary stones, atherosclerotic plaques, and dental calculus. Taken together, the present findings suggest that osteopontin produced by dental pulp cells is possibly associated with calcification of the pulp stone matrix.

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.

The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca2+‐permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT‐PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild‐type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up‐regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat‐induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.—Aijima, R., Wang, B., Takao, T., Mihara, H., Kashio, M., Ohsaki, Y., Zhang, J.‐Q., Mizuno, A., Suzuki, M., Yamashita, Y., Masuko, S., Goto, M., Tominaga, M., Kido, M. A., The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia. FASEB J. 29, 182–192 (2015). www.fasebj.org

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.

Establishment and characterisation of an osteoblastic clonal cell line from human mandibular osteosarcoma (HMOS-1)

A human mandibular osteosarcoma cell line, HMOS-1, with osteoblastic phenotypes and tumor-genicity was established. The cell line showed high alkaline phosphatase (ALP) activity immediately after seeding, with its peak around the 7th to 10th day of culture (1.44 mumol/min/mg protein). Parathyroid hormone (PTH) enhanced the ALP activity as well as intracellular cAMP production in a concentration-dependent manner. The effects of PTH on both ALP activity and cAMP production were expressed more strongly at the end stage of logarithmic cell growth than at the resting stage. 1,25 (OH)2D3 also stimulated the ALP activity, but its effect was low and was not different in any of the different culture stages. When these cells were transplanted to BALB/C nude mice, similar tumours to the original one, with abundant osteoids were observed. However, th e synthesis of type 1 collagen was not detected in the culture medium. The results indicate that the HMOS-1 cell line expresses an immature pre-osteoblastic phenotype. Because of these characteristics, HMOS-1 cells should be useful, not only in studies on the differentiated phenotypes of human osteoblasts, but also in studies on the diagnosis, treatment and aetiology of human osteosarcoma of the jaw.